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QP51 4  .Sa3  A  laboratory  manual 


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A  LABORATORY  MANUAL 

OF 

PHYSIOLOGICAL  AND  PATHOLOGICAL 
OHEMISTM. 

FOE  STUDENTS  IN  MEDICINE 


DE.    E.    SALKOWSKI 

Professor  in  the  University  and  Director  of  the   Chemical  Laboratory  of  the 
Pathological  Institute,  Berlin 


AUTHORIZED   TRANSLATION  FROM  THE  SECOND  REVISED   AND 
ENLARGED  GERMAN  EDITION 

BY 

^\.  R.  ORNDORFF,  A.B.,  Ph.D. 

Professor  of  Organic  and  Physiological  Chemistry  in  Cornell  University 


Xaitb  'Cen  ifigures  an^  a  Colored  plate  of  absorption  Spectra 


FIRST  EDITION 
FIRST   THOUSAND 


NEW   YORK 

JOHN  WILEY  &  SONS 

London:  CHAPMAN  &  HALL,   Limited 

1904 


Copyright,  1904, 

BY 

W,  R.  OENDORFF. 


aOBEBT  DBTJMMOND,  PRINTER,  VEW  YORK. 


DEDICATED   TO 

professor  1Ru&olf  Dircbow 

AS   A   TOKEN"    OF   THE    ESTEEM    AND    GRATITUDE    OF 
THE    AUTHOR. 


Digitized  by  tlie  Internet  Arcliive 

in  2010  witli  funding  from 

Open  Knowledge  Commons  (for  the  Medical  Heritage  Library  project) 


http://www.archive.org/details/laboratorymanualOOsalk 


PKEFACE  TO  THE  ENGLISH  EDITION. 


I  AM  very  much  pleased  that  Dr.  Orndorff  has  taken 
upon  himself  the  translation  into  English  of  the  second  edi- 
tion of  my  book  ''Practicum  der  physiologischen  und 
pathologischen  Chemie."  The  exact  knowledge  of  German 
which  Dr.  Orndorff  has  acquired  by  long  residence  in  Germany, 
together  with  his  well-known  scientific  qualifications,  guaran- 
tees a  correct  translation. 

That  part  of  the  book  dealing  with  inorganic  chemistry 
has  not  been  translated,  as  the  students  in  medicine  in 
America  have  usually  completed  a  required  course  in  quali- 
tative analysis  before  they  take  up  organic  and  physiological 
chemistry.  In  the  description,  moreover,  of  the  Kjeldahl 
method  of  determining  the  amount  of  nitirogen  in  organic 
substances,  the  modification  adopted  by  the  "Association 
of  Official  Agricultural  Chemists  of  the  United  States,"  which 
is  the  one  generally  used  in  America,  has  been  given.  It  has 
therefore  also  seemed  expedient  to  omit  the  Schneider- 
Seegen  method. 

A  number  of  errors  in  the  German  edition  have  been  cor- 
rected in  this  translation ;  and  some  additions  and  changes 
have  been  made,  those  due  to  Dr.  Orndorff  being  indicated 
in  foot-notes.  The  tables  of  specific  gravities  given  are  the 
most  accurate  that  have  been  published. 


vi  PREFACE  TO  THE  ENGLISH  EDITION, 

I  cherish  the  hope  that  the  book  in  this  new  form  will  con- 
tribute its  modest  share  towards  gaining  new  friends  abroad 
for  the  study  of  physiological  chemistry,  so  successfully  pur- 
sued in  America. 

Dr.  E.  Salkowski. 

Berlin,  Dec.  19,  1902. 


CONTENTS. 


CHAPTER  I. 

Ex,4MiNATiON  OF  Milk 

CHAPTER  II. 
Examination  of  Muscular  Tissue 20 

CHAPTER  III. 

Gastric  Digestion 

oo 


CHAPTER  X. 
Examination  of  the  Ukxnr 


48 


64 


70 


CHAPTER  IV. 

EXAinNATION   OF   BlOOD 

CHAPTER  V. 
Pathological  Transudates,  Cystic  Fluids 

CHAPTER  VI. 
Saliva  and  Salivaby  Digestion 

CHAPTER  VII. 
Examination  of  the  Pancreas ^g 

CHAPTER  VIII. 

Examination  of  Bile.  ...  or 

o5 

CHAPTER  IX. 
Examination  of  Biliart  Calculi 


90 


95 

vii 


viii  CONTENTS. 

CHAPTER  XI. 

PAGE 

Examination  op  Urinary  Calculi.  ,  .     128 

CHAPTER  XII. 
Examination  of  the  Liver. 131 

CHAPTER  XIII. 
Examination  of  Bone 137 

CHAPTER  XIV. 
Examination  of  Adipose  Tissue 141 

CHAPTER  XV. 
Yolk  and  White  of  the  Egg 1 50 

CHAPTER  XVI. 
The  Products  op  the  Putrefaction  of  Proteids 159 


QUANTITATIVE  ANALYSIS. 
I. 

Quantitative  Analysis  op  Some  Inorganic  Compounds 175 

II. 

Analysis  of  the  Urine ISO 

III. 
Analysis  op  the  F^ces.. 211 

IV. 
Analysis  op  Meat 217 

V. 
Analysis  of  Milk 221 

VI. 
Analysis  op  Bread 229 


CONTENTS.  IX 

VII. 

PAGE 

Analysis  op  Blood 231 

VIII. 

Determixatiox  of  Hydrochloric  Acid  in  the  Gastric  Juice  ....   237 

IX. 

Quantitative  Digestion  Experiments 240 

X. 

Determination  of  Glycogen 244 

APPENDIX  I. 
List  of   Reagents 249 

APPENDIX  II. 
Tables  of  Specific  Gravities 253 

APPENDIX  III. 
International  Atomic  Weights 257 

Index 259 

Index  to  the  Colored  Plate  of  Absorption  Spectra 264 


ABBREVIATIONS. 

cm.  for  centimeter.         cc.  for  cubic  centimeter, 
mm.  for  millimeter.        g.  for  gram. 
1.  for  liter.  mg.  for  milligram, 

kg.  for  kilogram. 


A  LABOEATORY  MANUAL 

OF 

PHYSIOLOGICAL  AND  PATHOLOGICAL 
CHEMISTET. 


CHAPTER  I. 

EXAMINATION  OF  MILK. 

I.  General  Properties  of  Milk. 
II.  Separation  of  Milk  into  its  Constituents. 

III.  Precipitation  with  Magnesium  Sulphate. 

IV.  Action  of  Rennin  on  Milk. 
V.  Preparation  of  Lactic  Acid. 

I.  GENERAL  Properties. 

1.  The  reaction  of  milk  is  usually  amphoteric,  i.e.,  it  red- 
dens blue  litmus  paper,  turns  red  litmus  paper  blue,  and  does 
not  act  on  violet  litmus  paper.  Towards  phenol-phthalein 
it  reacts  acid,  towards  lacmoid  alkaline. 

2.  On  heating  fresh  milk  to  boiling,*  it  does  not  coagulate, 
its  general  appearance  is  not  changed.  When  heated  for  a 
longer  time  it  forms  a  skin  on  the  surface  consisting  essen- 

'  All  reactions  or  tests  are  to  be  performed  in  test-tubes  unless  other- 
wise directed. 


2       PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

tially  of  evaporated  milk.  The  specific  odor  of  milk  becomes 
stronger  on  heating.  Milk  which  is  a  few  days  old  coagulates 
on  heating. 

3.  On  the  addition  of  acids,  milk  coagulates,  the  casein 
being  precipitated. 

4.  When  equal  volumes  of  milk  and  caustic  soda  solution 
are  heated  to  boiling,  the  mixture  becomes  yellow  and  finally 
brown,  owing  to  the  action  of  the  caustic  soda  on  the  milk- 
sugar. 

5.  If  we  shake  a  few  cubic  centimeters  of  milk  with  one 
and  one-half  to  two  times  its  volume  of  ether,  the  appearance 
of  the  milk  is  only  shghtly  changed,  though  the  fat  is  for  the 
most  part  extracted.  In  order  to  show  this,  pour  a  part  of 
the  ether  into  a  watch-glass,  taking  care  that  none  of  the  milk 
gets  into  the  glass.  On  evaporating  in  the  air  the  ether 
leaves  the  fat  behind.  If  we  now  pour  some  caustic  soda 
solution  down  the  side  of  the  tube  and  mix  the  contents  by 
shaking  gently,  the  milk  becomes  almost  clear.  This  shows 
that  the  white  color  of  milk  is  due  only  in  small  part  to  the 
fat  which  it  contains;  to  a  much  greater  extent  it  is  due  to 
the  swollen  casein  (and  calcium  phosphate). 

6.  When  some  tincture  of  guaiacum  (made  by  dissolving 
some  gxmi  guaiacum  in  alcohol  in  a  test-tube)  is  added  to  a 
little  milk,  then  some  old  oil  of  turpentine,  and  the  mixture 
shaken,  it  becomes  blue.  The  color  appears  first  at  the  sur- 
face of  contact  of  the  milk  and  the  oil  of  turpentine  (Kowa- 
lewsky).  Boiled  milk  does  not  show  this  reaction.  Milk, 
blood  or  blood-pigment,  and  the  pus-cells  possess  this  prop- 
erty in  common.  To  distinguish  boiled  milk  from  unboiled, 
the  following  method  of.Schaffer  is  also  recornmended:  add 
to  10  cc.  of  milk  one  drop  of  a  0.2  per  cent,  solution  of  hydro- 
gen peroxide  and  two  drops  of  a  2  per  cent,  solution  of  para- 
phenylene  diamine.  Fresh  milk  turns  blue,  boiled  milk  does 
not. 


EXAMINATION  OF  MILK. 


II.  Separation  of  Milk  into  its  Constituents. 

Milk,  diluted  with  water,  precipitated  AA-ith  acetic 
acid  and  filtered. 


Residue  on  filter  (A)  casein  and  Filtrate  (B),  albumin,  milk-sugar, 

fat;  extracted  with  ether.  salts;  evaporated  at  the  boil- 

ing-point. 


Residue :  casein      Solution  evapo-    Coagulated  albu-  Evaporated  fur- 
with  some  fat  rated:  butter-       min  (E).  ther:  calcium 

(C).  fat(D).  phosphate    (F), 

milk-sugar  (G). 

Dilute  400  cc.  of  milk  (whole  milk)  in  a  beaker  with  1  liter 
of  water,  and  then  add  acetic  acid/  cautiously  with  stirring, 
until  the  casein  separates  in  coarse  flakes.  An  excess  of  acetic 
acid  is  to  be  carefully  avoided.  The  operation  may  be  made 
easier  by  taking  out  small  amounts  of  the  milk  in  a  beaker, 
adding  acetic  acid,  and  observing  whether  the  casein  becomes 
more  coarsely  flocculent.  The  casein  incloses  the  fat  com- 
pletely and  carries  it  down  with  it.  The  action  of  the  acetic 
acid  is  to  remove  the  alkaU  by  means  of  which  the  casein  is 
dissolved  in  the  milk.  The  mixture  is  filtered  through  mus- 
lin, or  the  clear  supernatant  fluid  is  siphoned  off  and  only  the 
residue  filtered. 

The  mixture  (A)  of  casein  and  fat  remaining  on  the  filter 
is  washed  once  with  water,  drained,  and  the  filter  is  lightly 
squeezed  with  the  hand  to  remove  the  excess  of  wash-water. 
It  is  then  ground  in  a  mortar  with  100  cc.  of  absolute  alco- 
hol, which  takes  up  the  greater  part  of  the  water  and  some 
of  the  fat,  and,  after  standing  for  half  an  hour,  filtered.  The 
alcohol  is  evaporated  in  a  porcelain  dish  on  the  water-bath 
and  the  dish  with  the  residue  set  aside.    To  separate  the 

'  Thirty  per  cent,  acid,  sp.  gr.  1.041,  is  always  understood  by  this  term. 
See  table  of  reagents  at  the  end  of  the*  book.         - — 


4       PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

casein  and  fat  put  the  casein  containing  the  fat  into  a  dry 
flask  with  about  100  cc.  of  ether,  shake  thoroughly,  allow  to 
stand  twenty-four  hours,  and  then  filter. 

The  casein  remaining  on  the  filter  (C)  is  washed  once  more 
with  ether,  then  pressed  between  filter-paper,  and  ground  in 
a  mortar  until  the  casein,  still  containing  some  fat,  forms  a 
white  dry  powder. 

The  ethereal  solution  (D)  is  poured  into  the  evaporating- 
dish  in  which  the  alcohol  extract  was  evaporated  and  the 
solution  allowed  to  evaporate  spontaneously,  taking  care  that 
the  ether  vapor  does  not  come  into  contact  with  a  flame. 
Finally  it  is  separated  from  a  small  residue  of  water,  alcohol, 
and  ether  by  evaporating  on  the  water-bath.  Thus  we  ob- 
tain the  butter-fat. 

The  fluid  (B)  separated  from  the  casein  is  filtered  through 
paper  and  then  evaporated  about  one-half  at  the  boiling- 
point  in  a  tinned  vessel  or  in  an  enamelled-iron  dish.  The 
albumin  (E)  precipitates  in  coarse  white  flakes ;  it  is  filtered 
off  and  washed  a  few  times  with  hot  water.  The  filtrate  from 
the  albumin  is  evaporated  further  over  a  free  flame  until  it 
begins  to  bump.  The  bumping  is  due  to  the  separation  of 
calcium  phosphate  (F).  Filter  once  more  and  evaporate  on 
the  water-bath;  the  sirupy  solution  yields  an  abundant  crop 
of  milk-sugar  crystals  when  allowed  to  stand  till  next  day. 

Properties  and  Reactions  of  the  Constituents  of  Milk. 
I.  Coagulated  Albumin  (E). 

GENERAL  REACTIONS  OF  THE  COAGULATED  ALBUMINS. 

1.  Xanthoproteic  Reaction.  A  portion  of  the  substance 
the  size  of  a  pea  is  heated  in  a  test-tube  with  concentrated 
nitric  acid  (sp.  gr.  1.2).  The  albumin  turns  yellow  and  at  the 
same  time  a  yellow  solution  is  formed.  When  perfectly  cold 
supersaturate  with  caustic  soda  solution.    The  color  turns  to 


/ 

EXAMINATION  OF  MILK.  5 

orange  and  the  undissolved  particles  of  albumin  also  take  on 
the  same  color.  This  reaction  depends  upon  the  conversion 
of  the  aromatic  group  in  the  albumin  molecule  into  nitro 
derivatives. 

2.  Millon's  Reaction.  Treat  a  small  portion  of  the  sub- 
stance vnth  a  few  cubic  centimeters  of  water,  add  some  some 
IVIillon's  reagent,  and  heat  to  boiUng.  The  albumin  turns 
brick-red.  This  reaction  is  due  to  the  tyrosin  group  present 
in  the  albumin  molecule ;  tyrosin  gives  the  reaction  in  a  strik- 
ing manner,  and  the  same  is  true  of  all  derivatives  of  benzene 
in  which  a  benzene  hydrogen  atom  is  replaced  by  hydroxyl. 
(0.  Nasse.) 

3.  Conduct  towards  an  Alkaline  Solution  of  Lead  Hydroxide. 
To  some  cubic  centimeters  of  caustic  soda  solution  add  two 
drops  of  a  neutral  lead  acetate  solution.  The  precipitate  of 
lead  hydroxide  first  formed  dissolves  on  shaking.  Heat  a 
small  portion  of  the  albumin  with  this  alkaUne  solution  of 
lead  hydroxide;  the  mixture  turns  black  in  consequence  of 
the  formation  of  lead  sulphide.  A  part  of  the  sulphur  is 
present  in  albumin  in  the  unoxidized  form  and  may  be  spUt 
off  by  alkahes  as  potassium  or  sodium  sulphides, 

4.  Reaction  of  Adamkiewicz.  Grind  a  part  of  the  albumin 
in  a  small  mortar  with  some  absolute  alcohol,  filter,  squeeze 
out  the  excess  of  alcohol,  and  grind  with  some  cubic  centi- 
meters of  ether,  filter,  and  remove  the  excess  of  ether  by  pres- 
sure. Use  half  of  the  albumin  thus  obtained  for  the  reaction 
of  Adamkiewicz;  dissolve  the  albimiin  by  warming  with  a 
few  cubic  centimeters  of  glacial  acetic  acid,  cool  the  solution, 
(by  dipping  into  water),  and  allow  some  concentrated  sul- 
phuric acid  to  flow  slowly  down  the  wall  of  the  test-tube  into 
the  acetic  acid  solution  of  the  albumin:  the  fluids  mix  at  the  ' 
surface  of  contact,  giving  a  violet  to  purple  color. 

According  to  Hopkins  and  Cole  ^  this  reaction  is  due  to 

'  Proc.  Roy.  Soc,  68,  21  (1901).  ^~ 


Yv 


6       PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

the  presence  of  glyoxylic  acid  in  the  glacial  acetic  acid.  If 
the  reaction  does  not  take  place,  add  a  small  amount  of  a 
solution  of  oxahc  acid  which  has  been  previously  treated  with 
sodium  amalgam. 

5.  Liebermann's  Reaction.  Add  to  the  other  half  of  the 
albumin,  treated  with  alcohol  and  ether,  a  few  cubic  centi- 
meters of  fuming  hydrochloric  acid  and  warm :  bluish  to  blue 
solution,  which,  on  standing,  becomes  paler  or  more  violet 
or  brownish.     Sometimes  only  the  violet  color  appears. 

6.  Detection  of  Nitrogen.  A  small  portion  of  the  albumin, 
which  has  been  previously  treated  with  alcohol  and  ether  and 
then  dried,  is  mixed  with  five  to  ten  times  its  volume  of  soda- 
lime  and  the  mixture  heated  in  a  narrow  dry  test-tube.  Am- 
monia is  given  off  (odor;  alkaUne  reaction  of  the  gas;  fumes 
which  it  forms  when  brought  into  contact  with  a  glass  rod 
moistened  with  hydrochloric  acid;  blackening  of  a  piece  of 
filter-paper  moistened  with  a  solution  of  mercurous  nitrate.) 

The  test  for  nitrogen  with  soda-hme  fails  in  the  case  of 
certain  forms  of  combination  of  nitrogen,  e.g.,  with  the  nitro 
compounds.  Of  more  general  application  and  also  more 
delicate  is  the  Lassaigne  Test.  Heat  the  substance  with  a 
small  piece  of  potassium  (or  sodium)  in  a  narrow  test-tube. 
A  violent  reaction  takes  place.  After  the  test-tube  has 
cooled  somewhat,  dip  it  into  10  cc.  of  water  contained  in  a 
small  beaker;  the  test-tube  breaks  and  its  contents  dissolve 
in  the  water.  When  nitrogen  is  present  the  solution  will  con- 
tain potassium  cyanide.  Filter  the  solution,  add  a  drop  of 
ferric  chloride  solution  and  a  few  drops  of  ferrous  sulphate 
solution  to  the  filtrate  and  warm.  The  potassium  cyanide 
forms  potassium  ferrocyanide.  Cool  the  solution  and  acidify 
with  hydrochloric  acid.  Green  or  blue  color  or  a  blue  pre- 
cipitate due  to  the  formation  of  Prussian  blue. 

7.  Detection  of  Sulphur.  1.  Heat  a  very  small  piece  of 
albumin  in  a  narrow  hard-glass  test-tube,  the  upper  part  of 


EXAMINATION  OF  MILK.  7 

■which  contains  a  narrow  strip  of  filter-paper  moistened  with 
a  solution  of  basic  lead  acetate;  blackening  due  to  the  forma- 
tion of  lead  sulpliide  (Siegfried).  2.  Grind  together  0.1  to 
0.2  g.  of  the  substance  with  tliirty  times  its  weight  of  oxidiz- 
ing mixture  (3  parts  of  potassium  nitrate  and  1  part  of  sodium 
carbonate),  and  heat  the  mixture  slowly  in  a  crucible  or  small 
dish  until  it  is  completely  fused  and  all  the  carbon  has  been 
burned.  The  sulphur  is  oxidized  to  sulphuric  acid,  which 
forms  alkaU  sulphate.  After  cooling  dissolve  the  fused  mass 
by  warming  with  some  water,  filter  in  case  the  solution  is  not 
perfectly  clear,  acidify  with  hydrochloric  acid,  and  add  barium 
chloride  solution.  If  the  substance  contained  sulphur,  a  pre- 
cipitate of  barium  sulphate  will  separate  at  once,  or  after  some 
time.  Since  the  test  depends  upon  the  conversion  of  sulphur 
into  sulphuric  acid,  the  substance  and  the  reagents  must  of 
course  contain  no  sulphates. 

This  method  is  not  adapted  to  detect  traces  of  sulphur:  in  this 
case  we  must  evaporate  the  solution  of  the  fused  mass  to  dryness 
.several  times  with  hydrochloric  acid  in  order  to  drive  out  the  nitric 
acid.  If  we  then  add  water  to  the  residue,  we  sometimes  find  the 
solution  is  cloudy,  due  to  the  presence  of  silicic  acid.  It  must  then 
be  filtered  again,  since  clearness  of  the  solution  is  absolutely  essential. 
The  sulphur  may  also  be  detected  by  heating  to  red  heat  with  sodium 
(or  potassium),  sodium  sulphide  being  formed.  In  many  cases  it  is 
sufficient  tc  heat  with  sodium  carbonate.  See  in  this  connection  the 
*' Detection  of  Sodium  Sulphide"  in  the  chapter  on  "Bile,"  section 
^'Taurin." 

2.  Calcium  Phosphate  (F). 

The  calcium  phosphate  is  washed  with  water,  then  dis- 
solved by  pouring  on  the  filter  20  cc.  of  dilute  hydrochloric 
acid  (1  part  hydrochloric  acid,  2  parts  water).  The  filtrate 
is  frequently  somewhat  cloudy,  but  it  may  be  rendered  clear 
by  allowing  it  to  stand  and  by  filtering  several  times,  or  also 
by  repeatedly  pouring  it  back  upon  the  filter.    The  greater 


8       PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

part  of  the  filtrate  is  made  alkaline  with  ammonia,  acidified 
with  acetic  acid  and  then  divided  into  two  parts ;  to  one  part, 
in  order  to  prove  the  presence  of  calcium,  add  ammonium 
oxalate  (white  precipitate  of  calcium  oxalate);  to  the  other 
part  add  uranyl  nitrate  (yellowish-white  precipitate  of  uranyl 
phosphate).  The  phosphoric  acid  may  also  be  tested  for 
directly  in  the  hydrochloric  acid  solution  by  means  of  ammo- 
nium molybdate.  Add  a  few  drops  of  the  hydrochloric  acid 
solution  to  some  cubic  centimeters  of  the  ammonium  molyb- 
date solution:  yellow  precipitate,     i^    11?^  ^>/ 

3.  Casein  (C). 

In  order  to  purify  the  casein  and  especially  to  free  it  from 
the  fat,  put  it  into  a  dish  with  250  cc.  of  water  and  add  very 
dilute  caustic  soda  solution  (1  :  10)  with  constant  stirring  and 
quite  slowly.  The  mixture  must  at  no  time  have  a  strong 
alkahne  reaction.  When  the  greater  part  of  the  casein  has 
dissolved,  filter.  The  filtrate  is  usually  somewhat  cloudy. 
If  necessary  it  maybe  again  filtered;  absolute  clearness  of  the 
filtrate  is,  however,  only  attained  with  difficulty.  The  solu- 
tion is  precipitated  by  acidifying  cautiously  with  acetic  acid. 
The  casein  which  separates  is  first  washed  by  decantation, 
which  may  usually  be  done  without  any  essential  loss,  then 
filtered  and  washed. 

This  is  used  in  the  following  reactions: 

1.  Since  casein  is  essentially  a  proteid,  it  gives  all  the 
reactions  of  the  coagulated  and  insoluble  proteids  described 
under  albumin. 

2.  A  portion  is  shaken  with  water  and  a  few  drops  of 
sodium  carbonate  solution;  it  dissolves  therein  clear  or  almost 
clear.  If  the  solution  is  quite  cloudy  (fat,  calcium  phosphate), 
then  the  casein  must  be  redissolved  in  water  containing 
sodium  hydroxide  and  be  precipitated  once  more  with  acetic 
acid. 


EXAMINATION  OF  MILK.  9 

3.  A  portion  is  ground  \\ith  water  and  some  calcium  car- 
bonate, and  filtered.  The  filtrate,  which  is  usually  not  quite 
clear,  contains  casein,  as  may  be  shown  by  acidifying  with 
acetic  acid.  Casein  therefore  has  the  character  of  an  acid: 
it  drives  out  the  carbonic  acid  and  forms  a  soluble  salt  with 
calcium. 

4.  With  a  portion  of  the  substance  try  the  reaction  with 
the  alkaUne  solution  of  lead  hydroxide  described  under  Albu- 
min. Only  a  faint  gray  color  results.  Casein  contains  only 
a  httle  of  the  unoxidized  sulphur ;  the  greater  part  is  in  the 
oxidized  form.  In  order  to  show  more  exactly  the  difference 
between  casein  and  albumin  in  this  reaction,  we  proceed  as 
follows:  the  solution  of  caustic  soda  containing  the  lead  is 
diluted  mth  several  times  its  volume  of  water  and  the 
dilution  is  continued  until  some  of  the  diluted  solution 
when  boiled  with  albumin  is  only  sHghtly  blackened.  The 
casein  is  then  tested  with  this  solution.  It  should  react 
negatively. 

5.  Casein  is  not  a  simple  proteid,  but  may  be  spUt  up  by 
appropriate  means  (digestion  in  the  stomach)  into  a  proteid 
and  paranuclein.  Since  paranuclein,  like  nuclein  itself,  con- 
tains organically  combined  phosphorus,  the  casein  also 
contains  phosphorus.  In  order  to  prove  the  presence  of 
phosphorus,  we  grind  about  0.2  g.  of  the  casein,  which  has 
pre\'iously  been  treated  with  alcohol  and  ether,  with  6  g.  of 
the  oxidizing  mixture  (see  above  under  Detection  of  Sulphur), 
heat  to  fusion,  dissolve  the  fused  mass,  after  coohng,  in 
nitric  acid  and  heat  the  solution  in  order  to  drive  out  the 
nitrous  acid  formed.  A  part  of  the  solution  is  added,  drop 
by  drop,  to  about  5  cc.  of  the  molybdate  solution;  yellow 
color,  cloudiness,  and  then  a  yellow  precipitate  prove  the 
presence  of  phosphoric  acid,  formed  from  the  phosphorus  by 
the  fusion  with  the  niter.  The  reaction  is  only  to  be  relied 
on  to  prove  the  presence  of  phosphorus  when  the  substance 


10     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

is  free  from  calcium  or  magnesium  phosphate.^  We  deter- 
mine this  by  adding  ammonia  to  the  rest  of  the  solution.  It 
must  remain  clear. 

4.  Butter-fat  (D). 

The  butter-fat  is  saponified.  All  heating  and  evaporating 
in  this  section  is  to  be  done  on  the  water-bath.  Put  5  g.  of 
caustic  potash  into  a  flask,  add  5  cc.  of  water,  and  dissolve 
the  potash  by  warming.  Then  melt  the  butter-fat  and  pom- 
it  into  the  flask.  Wash  the  dish  with  about  50  cc.  of  90  per 
cent,  alcohol,  add  the  alcohol  to  the  flask,  and  heat  the  mix- 
ture, with  constant  shaking,  until  it  becomes  homogeneous. 
The  fat  is  thus  spht  up  into  fatty  acids  and  glycerin,  saponi- 
fied. In  order  to  determine  whether  the  saponification  is 
complete,  pour  a  small  portion  of  the  mixture  into  a  httle 
water;  it  must  form  a  clear  solution  or  one  which  becomes 
clear  on  gently  warming.  If  it  does  not  do  this  the  mixture 
must  be  again  heated.  When  the  saponification  is  completed 
pour  the  contents  of  the  flask  into  an.  evaporating-dish,  drive 
off  the  alcohol  by  heating  (water-bath),  and  when  cold  acidify 
with  30  cc.  of  dilute  sulphuric  acid.  The  fatty  acids  separate 
in  the  form  of  an  oil,  while  at  the  same  time  the  odor  of  butyric 
acid  becomes  perceptible,  due  to  the  presence  of  butyrin  in 
the  milk-fat.  This  butyrin  is  characteristic  of  milk-fat.  It 
is  only  found  in  this  substance. 

The  more  detailed  investigation  of  fat  and  the  fatty  acids 
will  be  taken  up  in  the  chapter  on  "Subcutaneous  Adipose 
Tissue." 

5.  Milk-sugar,  C12H22O11+H2O. 

The  milk-sugar  (G)  is  separated  from  the  mother-liquor 
by  draining  and  pressing  between  filter-paper  and  is  recrys- 

^  Alkali  phosphates  could  not  be  present  owing  to  the  previous  treat- 
jnent. 


EXAMINATION  OF  MILK.  H 

tallized  from  hot  water.  For  this  purpose  put  the  milk- 
sugar  into  a  flask,  dissolve  it  by  heating  with  water  (some 
calcium  phosphate  always  remains  undissolved),  add  a  little 
bone-black  to  decolorize,  and  filter  hot.  Evaporate  the  solu- 
tion to  about  25  or  30  cc.  (to  sirupy  consistency)  on  the  water- 
bath,  let  stand  till  next  day,  and  place  the  crystals  which 
have  separated  on  drying-paper. 

The  milk-sugar  forms  hard,  shining  crystals,  which  dis- 
solve in  six  parts  of  cold  water,  more  readily  in  hot  water, 
and  only  sUghtly  in  alcohol. 

REACTIONS   OF  MILK-SUGAR. 

1.  A  small  quantity  heated  on  a  crucible-cover  or  on 
platinum-foil  turns  brown,  gives  off  the  odor  of  caramel, 
carbonizes,  and  finally  burns  completely,  leaving  very  little 
ash. 

Milk-sugar  Uke  grape-sugar  is  oxidized  in  alkahne  solu- 
tion; a  number  of  sugar  reactions  depend  upon  this  property. 
For  the  following  reactions  use  a  solution  of  2  g.  in  100  cc. 
of  water  and  another  solution  10  times  as  dilute  (10  cc.  of  the 
solution  diluted  to  100  cc). 

2.  Trommer's  Test.  To  a  few  cubic  centimeters  of  the 
solution  add  half  the  volume  of  caustic  soda  solution  (of 
about  1.17  sp.  gr.),  then  add  copper  sulphate  solution,  drop 
by  drop,  shaking  the  tube  after  the  addition  of  each  drop. 
A  deep-blue  solution  results,  which  on  heating  gives  a  pre- 
cipitate of  red  cuprous  oxide  (or  yellow  cuprous  hydroxide). 
This  property  of  dissolving  cupric  hydroxide  in  alkaline  solu- 
tion is  common  to  milk-sugar,  glucose,  and  to  many  other 
organic  substances,  such  as  cane-sugar,  glycerin,  mannite, 
and  tartaric  acid;  but  on  warming  these  solutions  no  reduc- 
tion takes  place,  except  in  the  case  of  milk-sugar  and  glucose. 

3.  Moore's  Test.  On  the  addition  of  an  equal  volume  of 
caastic  soda  solution  of  specific  gravity  1.34  and  heating  to 


12     PHYSIOLOCICAL  AND  PATHOLOGICAL  CHEMISTRY. 

boiling,  the  solution  turns  yellow,  then  brown,  and  develops 
the  odor  of  caramel,  especially  after  acidifying  with  dilute 
sulphuric  acid. 

4.  Bismuth  Test.  Saturate  some  of  the  solution  at  the 
boiUng-point  with  sohd  sodium  carbonate,  add  a  httle  bis- 
muth subnitrate,  heat  to  boiling,  and  keep  boihng  for  some 
time:  gray  or  black  color  due  to  the  formation  of  finely 
divided  metallic  bismuth.  This  reaction  may  also  be  per- 
formed with  sodium  hydroxide  instead  of  sodium  carbonate, 
using  only  a  very  sUght  amount  of  caustic  soda,  but  the 
simultaneous  action  of  the  alkaU  on  the  milk-sugar  cannot 
be  excluded  and  the  color  is  then  a  dirty  grayish  green. 

5.  To  a  few  cubic  centimeters  of  the  solution  add  some 
sodium  carbonate  solution  and  a  httle  freshly  prepared 
potassium  ferricyanide  solution.  Decolorization  on  warm- 
ing, due  to  the  formation  of  ferrocyanide  of  potassium. 

6.  To  some  of  the  solution  add  silver  nitrate  solution  and 
ammonia  and  warm.  Separation  of  metalhc  silver  in  the 
form  of  a  bright  mirror  or  a  gray  powder.  A  very  pretty 
mirror  is  obtained  if  we  use  an  excess  of  caustic  soda  with 
only  a  httle  ammonia  in  the  reaction  (caution  on  account  of 
the  possible  formation  of  fulminating  silver).  Cane-sugar 
and  mannite  also  give  the  reaction  imder  these  conditions, 
but  not  with  ammonia  and  silver  nitrate. 

7.  Indigo  Test.  Use  only  the  weaker  solution  of  the 
milk-sugar  in  this  test.  To  a  small  portion  of  the  sugar 
solution  add  some  freshly  prepared  solution  of  indigo  carmine 
or  sodium  indigo  sulphonate  until  a  blue  color  is  produced, 
make  alkahne  with  a  few  drops  of  sodium  carbonate  solution, 
and  warm.  The  solution  turns  first  violet,  then  red,  then 
yellow,  and  finally  becomes  almost  colorless.  The  reaction 
depends  on  the  reduction  of  indigo  blue  to  indigo  white. 
Pour  half  of  the  solution  into  another  test-tube  and  shake 
thoroughly  with  air.     It  turns  blue  again:   oxidation  of  the 


EXAMINATION  OF  MILK.  13 

indigo  white  to  indigo  blue.  Heat  again  and  the  solution 
will  be  again  decolorized.  Tliis  process  may  be  repeated 
until  all  the  sugar  is  used  up  by  oxidation. 

All  these  reactions  are  given  by  glucose  as  well  as  by  milk- 
sugar.  The  conduct  towards  yeast  is  the  simplest  method 
of  cUstinguisliing  between  the  two  sugars.  Glucose  is  very 
quickly  converted  into  alcohol  and  carbon  dioxide  by  yeast, 
whereas  milk-sugar  is  not,  or  at  least  very  slowly  and  incom- 
pletely. In  order  to  carry  out  the  experiment  shake  a  quan- 
tity of  the  2  per  cent,  milk-sugar  solution  in  a  test-tube  with 
a  piece  of  compressed  yeast  as  large  as  a  hazel-nut,  fill  a  fer- 
mentation-tube mth  the  mixture  (mercury  seal),  and  put  the 
tube  in  a  warm  place  (about  35°).  Make  a  similar  test  with 
a  2  per  cent,  solution  of  glucose  as  a  check.  After  some 
hours  the  glucose  solution  will  be  found  in  fermentation,  as 
shown  by  the  development  of  carbon  dioxide,  which  fills  a 
part  of  the  tube;  the  milk-sugar  solution  does  not  ferment. 
It  is  advisable  to  set  up  a  third  tube,  which  contains  only 
water  and  yeast.  No  fermentation  should  take  place  in  it 
during  twelve  to  twenty-four  hours.  Gradually  a  sUght 
development  of  carbon  dioxide  makes  itself  apparent  (spon- 
taneous fermentation  of  yeast). 

The  following  tests  may  also  be  used  to  distinguish  between  the 
two  sugars. 

1.  Rubner's  Test.  Dissolve  4  g.  of  neutral  lead  acetate  by  warm- 
ing with  about  5  cc.  of  the  2  per  cent,  solution  of  milk-sugar,  boU  for 
one  to  two  minutes,  then  add  an  excess  of  ammonia  and  heat  again. 
A  deep-red  solution  will  be  formed  and  gradually  a  precipitate  of  the 
.same  color  appears  if  sufficient  ammonia  has  been  added.  Glucose 
acts  in  the  same  way  at  first,  but  the  color  soon  becomes  yellow 
(chamois). 

Perhaps  the  following  slight  modification  of  the  test  is  somewhat 
simpler:  mix  about  3  oc.  of  milk-sugar  solution  (2  per  cent.)  with  the 
same  volume  of  basic  lead  acetate  solution  and  1  cc.  of  ammonia,  and 
boil  for  some  time.  The  milky  fluid  becomes  first  yellow,  then  brick- 
red,  and  then  remain.s  unchanged,  further  addition  of  'ammonia  in- 


14     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

ceasing  the  color.  Glucose  solution  (2  per  cent.),  when  treated  in 
the  same  way,  though  it  does  not  become  milky  at  first,  also  yields  a 
red  color  and  more  quickly  than  the  mUk-sugar;  the  precipitate,  how- 
ever, soon  becomes  yellow.  If  more  ammonia  be  added  (2  cc.)  there 
will  be  formed  at  first  a  very  beautiful  cherry-red  color,  which,  how- 
ever, also  disappears  very  quickly. 

2.  The  Formation  of  Mucic  Acid,  CeHioOg.  Five  grams  of  mUk-sugar 
are  put  into  a  flask  with  15  cc.  of  nitric  acid  of  specific  gravity  1.2,  and 
5  cc.  of  nitric  acid  of  about  1.48  specific  gravity,^  and  the  mixture  is 
cautiously  heated  untU  the  beginning  of  a  violent  reaction  (strong; 
evolution  of  oxides  of  nitrogen).  Remove  the  flame  at  once  and  let 
stand  tUl  next  day.  Pour  off  the  nitric  acid  from  the  crystallized 
mucic  acid,  wash  with  water,  first  by  decantation,  then  on  the  filter,. 
and  let  it  dry  on  filter-paper.  To  identify  the  substance  dissolve  a 
portion  of  the  mucic  acid  in  an  excess  of  ammonia,  evaporate  the 
solution  to  dryness  on  the  water-bath,  and  heat  the  dry  residue  in  a 
dry  test-tube:  pyrrol,  C^H^NH,  is  formed.  The  vapor  colors  a  pine 
splinter,  moistened  with  strong  hydrochloric  acid  and  held  in  the 
mouth  of  the  tube,  a  deep-red.  If  we  treat  glucose  in  the  same  way 
with  nitric  acid  nothing  separates.  The  solution  contains  a  con- 
siderable quantity  of  oxalic  acid,  but  no  mucic  acid. 

3.  The  Conduct  of  Milk-sugar  on  Heating  with  Hydrochloric  Acid» 
Dissolve  about  6  g.  of  commercially  pure  mUk-sugar  in  125  cc.  of 
water  by  heating  to  boiling,  let  cool,  and  determine  how  much  this 
solution  rotates  the  plane  of  polarized  light.  To  100  cc.  of  this  solu- 
tion in  a  flask  add  10  cc.  of  hydrochloric  acid  and  heat  for  half  an 
hour;  nearly  neutralize  the  solution  with  dilute  caustic  soda  (com- 
plete neutralization  woiild  make  the  color  of  the  solution  too  dark), 
let  cool,  pour  into  a  100-cc.  measiiring-flask  or  into  a  measm-ing-cylin- 
der,  fill  up  to  100  cc,  mix  thoroughly,  and  again  determine  the  amount 
of  rotation.  It  will  be  found  to  have  increased  (hydrolysis  of  the 
mUk-sugar  into  glucose  and  galactose,  which  latter  rotates  more  to 
the  right  than  lactose).  When  glucose  is  treated  in  the  same  way^ 
its  rotation  remains  unchanged  or  decreases  a  little,  in  consequence 
of  the  formation  of  humin  substances. 

-  Or,  instead  of  the  mixture,  20  cc.  of  nitric  acid,  1.3  specific  gravity. 


EXAMINATION  OF  MILK.  15 

III.  Precipitation  with  magnesium  Sulphate. 


Precipitate:  casein  (sodium)  Filtrate:  milk-sugar,  albumin, 

and  fat.  salts. 

Casein  may  also  be  precipitated  from  milk,  presumably 
in  combination  mth  alkali,  in  a  form  soluble  in  water  or 
dilute  salt  solutions.  The  precipitation  of  the  casein  in  this 
form  results  from  the  saturation  of  the  milk  with  cUfferent 
salts,  e.g.,  magnesium  sulphate.  Saturate  100  cc.  of  milk 
with  magnesium  sulphate  (about  50  g.  are  required)  by  shak- 
ing it  -^ith  the  finely  powdered  salt  in  a  flask.  When  the 
salt  is  entirely  or  almost  entirely  dissolved,  filter  through  a 
filter  moistened  with  a  saturated  solution  of  magnesium  sul- 
phate, and  wash  several  times  with  magnesium  sulphate 
solution.  The  precipitate  contains  casein  and  fat  besides  a 
globuhn-hke  body  (lactoglobuhn)  present  in  milk  in  very 
small  quantity.  In  the  filtrate  we  may  prove  the  presence 
of  albumin  by  heating  to  boiUng.  The  well-washed  and  still 
moist  precipitate  of  casein  and  fat  is  ground  in  a  mortar  with 
100  cc.  of  water,  when  the  casein  goes  into  solution.  The 
mixture  is  allowed  to  stand  till  next  day,  when  the  fat  will 
have  come  to  the  top ;  it  is  then  clarified  by  filtering  several 
times.  We  thus  obtain  a  Ught-bluish  opalescent  solution, 
scarcely  ever  entirely  clear,  which,  however,  may  be  used  to 
determine  the  amount  of  rotation.  The  solution  is  Isevo- 
rotatory.  On  the  addition  of  acetic  acid  the  casein  is  precipi- 
tated. 

IV.  Action  of  rennin  on  milk. 

(a)  Coagulation  of  Milk.  One-tenth  (0.1)  gram  of  the 
commercial  rennet  powder  is  dissolved  in  100  cc.  of  water. 
The  solution  is  usually  somewhat  cloudy,  but  it  may  be  used 


16    PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

without  filtering.^  100  to  200  cc.  of  milk^  are  heated  in  a 
beaker  to  40°,  5-10  cc.  of  the  rennin  solution  added  and  well 
stirred.  Coagulation  soon  takes  place.  The  coagulum,  con- 
sisting of  casein  and  fat,  gradually  separates,  leaving  a  fluid 
containing  albumin  and  sugar  (milk-serum  or  sweet  whey). 
The  process  is  perfectly  analogous  to  the  coagulation  of  blood 
with  the  formation  of  the  blood-clot  and  separation  of  the 
blood-serum.  The  casein  differs  somewhat  in  its  properties 
from  the  casein  precipitated  by  acids.  We  therefore  give  it 
the  name  paracasein  or  cheese.  Let  stand  till  next  day,  then 
pour  off  as  much  of  the  fluid  as  possible,  grind  the  coagulimi, 
the  cheese,  with  water,  filter,  wash  again  with  water,  and  press 
the  cheese  dry  between  folds  of  muslin.  In  order  to  remove 
most  of  the  fat  we  proceed  in  the  same  manner  as  given  under 
Casein.  When  washed  a  few  times  with  ether  the  paracasein 
is  almost  free  from  fat. 

The  paracasein  hke  the  casein  dissolves  readily  in  hme- 
water  as  well  as  in  water  to  which  sodium  hydroxide  or  sodium 
carbonate  solution  has  been  added.  It  is  reprecipitated  on 
the  addition  of  acetic  acid.  When  ground  with  water  and 
calcium  carbonate  the  paracasein  does  not  dissolve  as  readily 
as  the  casein.  The  paracasein  always  contains  calcium;  solu- 
ble calcium  salts  are  essential  for  the  coagulation  of  milk  by 
rennin  (Hammarsten).  The  sweet  whey  gives  the  guaiacum- 
turpentine  reaction,  and  when  heated  it  forms  a  voluminous 
precipitate  of  albumin. 

If  we  add  to  100  cc.  of  milk  5  cc.  of  a  1  per  cent,  solution 
of  sodium  oxalate,  then  the  rennin  ferment  and  heat  to  40°^ 
the  milk  does  not  coagulate  on  account  of  the  precipitation 
of  the  calcium  as  calcium  oxalate.     If,  however,  we  add  a 


^  The  solution  appears  to  be  more  active  when  twenty-four  hours  old 
than  when  freshly  prepared. 
^  Preferably  skimmed  milk. 


EXAMINATION  OF  MILK.  17 

small  quantity  of  calcium  chloride,  coagulation  takes  place 
at  once.^ 

(b)  Influence  of  Acids  and  Alkalies  on  the  Coagulation  of 
Milk  by  Rennin.  Into  each  of  three  test-tubes  A,  B,  and  C 
put  10  cc.  of  milk.  To  B  add  10  drops  of  dilute  hydrochloric 
acid  (1  cc.  of  hydrochloric  acid,  sp.  gr.  1.183,  to  150  cc.  of 
water).  No  separation  of  casein  should  take  place.  To  C 
add  1  to  2  drops  of  a  concentrated  solution  of  sodium  carbon- 
ate. To  A  add  neither  acid  nor  alkali.  To  each  of  the  three 
tubes  add  one-half  a  cubic  centimeter  or  10  drops  of  the 
rennin  solution  and  note  the  order  in  which  the  coagulation 
takes  place.  The  milk  in  B  coagulates  first,  then  that  in  A. 
The  milk  in  C  does  not  coagulate  at  all  or  coagulates  ex- 
tremely slowly.  Acids  aid  the  rennin  coagulation;  alkalies 
interfere  with  it  or  prevent  it  entirely.  The  coagulation  of 
milk  in  the  stomach  results  from  the  simultaneous  action  of 
the  hydrochloric  acid  and  the  rennin  ferment.  The  experi- 
ment may  also  be  performed  in  another  way.  Dilute  the 
rennin  solution  so  that  one-half  of  a  cubic  centimeter  or  10 
drops  of  it  will  just  bring  about  the  coagulation  of  100  cc. 
of  milk  in  ten  minutes  or  will  no  longer  coagulate  it.  Now 
add  to  10  cc.  of  milk  10  drops  of  the  dilute  hydrochloric  acid 
(0.25  per  cent.)  and  then  10  drops  of  the  diluted  rennin  solu- 
tion. Coagulation  results  before  the  expiration  of  ten  min- 
utes. 

V.   LACTIC  ACID  FERMENTATION.^ 

To  a  solution  of  50  g.  of  cane-sugar  in  500  cc.  of  water 
add  20  g.  of  precipitated  chalk  and  about  30  cc.  of  sour  milk, 
and  let  the  mixture  stand,  in  an  open  flask  or  one  loosely 

*  Arthus  and  Pag^s,  Arch,  de  Physiologie,  1891,  pp.  331  and  540. 

'  For  a  better  method  of  preparing  lactic  acid  see  Die  Zersetzung  stick- 
stofffreier  organLgchen  Substanzen  durch  Bacterien  von  Dr.  O.  Emmerling, 
p.  32.— O. 


18    PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

stoppered  with  cotton,  six  to  eight  days  at  a  temperature  of 
40°,  shaking  the  flask  frequently.  The  cane-sugar  will  be 
transformed  for  the  most  part  into  lactic  acid,  which  com- 
bines with  the  calcium.  After  the  lapse  of  the  time  given 
most  of  the  chalk  will  be  dissolved.  Boil  the  mixture,  filter, 
concentrate  to  a  small  volume  on  the  water-bath,  and  allow 
to  crystalHze.  The  crystallized  calcium  lactate  is  dried  by 
pressing  between  drying-paper  and  purified  by  recrystallizing 
from  hot  water,  using  some  bone-black  to  decolorize  if  neces- 
sary. The  calcium  salt  is  then  converted  into  the  zinc  salt. 
Determine  the  weight  of  the  air-dried  calcium  salt  and  weigh 
off  the  equivalent  quantity  of  zinc  sulphate  (to  308  parts  of 
calcium  lactate  287  parts  of  the  crystalUzed  zinc  sulphate) 
or  a  little  less.  Dissolve  each  salt  in  a  httle  water,  mix  the 
solutions,  filter,  after  allowing  to  stand  for  some  time,  from 
the  calcium  sulphate  which  separates,  evaporate  on  the 
water-bath,  let  stand  till  next  day,  and  purify  the  zinc  lac- 
tate crystals  obtained  by  recrystallization  (microscopic 
crystal  form).  From  the  zinc  lactate  we  obtain  the  free 
lactic  acid  by  conducting  hydrogen  sulphide  through  its  solu- 
tion until  the  zinc  is  completely  precipitated.  The  solution 
filtered  from  the  zinc  sulphide  is  evaporated  and  the  lactic 
acid  obtained  is  purified  by  dissolving  in  a  mixture  of  equal 
volumes  of  alcohol  and  ether  (small  quantity),  filtering  and 
evaporating. 

The  fermentation  lactic  acid,  CH3CHOHCOOH  (ordinary, 
inactive,  ethyhdene  lactic  acid),  forms  a  colorless  or  pale- 
yellow  sirupy  fiuid  of  strong  acid  reaction,  miscible  in  every 
proportion  with  water,  alcohol,  and  ether. 

For  the  lactic  acid  reaction  of  Uffelmann  see  the  chapter 
on  Digestion. 

A  part  of  the  lactic  acid  obtained  is  used  for  the  preparation  of 
pure  zinc  lactate.  Boil  with  water  and  an  excess  of  basic  zinc  car- 
bonate, filter,  and  evaporate  to  crystallization  (it  is  best  to  use  the 


EXAMINATION  GF  MILK.  19 

freshly  precipitated  basic  zinc  carbonate  made  as  follows :  2  g.  of  zinc 
sulphate  are  dissolved  in  about  100  cc.  of  water,  heated,  sodium  car- 
bonate solution  gradually  added  untU  the  reaction  is  markedly 
alkaline,  and  the  precipitate  washed  free  from  sulphates  and  sodium 
carbonate  with  hot  water,  first  by  decantation  and  then  on  the  filter) . 
The  well-pressed  air-dried  zinc  lactate,  (C3H503)2Zn  +  3H20,  contains 
3  molecules  of  water  of  crystallization  (18.17  per  cent.),  which  is  driven 
oflf  at  100°  to  110°.  The  detepmination  of  the  amount  of  water  of 
crystallization  serves  to  distinguish  it  from  the  zinc  sarcolactate, 
which  only  contains  2  molecules  of  water  (12.89  per  cent.).  Drying 
over  sulphuric  acid  instead  of  in  the  air  is  inadmissible,  since  a  part 
of  the  water  of  crystallization  is  lost.  Indeed  by  drying  for  fourteen 
days  over  sulphuric  acid  the  whole  of  the  water  may  be  given  off. 


CHAPTER  II. 

EXAMINATION  OF  MUSCULAR  TISSUE. 

I.  Preparation  of  Creatine,  Detection  of  the  Xanthine  Bases. 
II.  Detection  of  the  Proteids. 

III,  Preparation  of  the  Xanthine  Bases  or  Alloxuric  Bases. 

IV.  Preparation  of  Sarcolactic  Acid. 

I.  Preparation  of  Creatine — detection  of  the 
Xanthine  bases. 

Meat,  finely  ground,  digested  with  water,  filtered,  and  the  residue 
^  subjected  to  p^essuxe. 

Filtrate  heated  to  boiling  and  filtered.  Residue. 


Filtrate  precipitated  with  basic  Residue;  coagulated 

lead  acetate  and  filtered.  albumin. 


Filtrate  freed  from  lead  by  means  of  HjS,  Precipitate :  lead  phos- 

filtered,  and  the  filtrate  evaporated;  creatine.  phate,  chloride,  and 

sulphate. 

Four  hundred  grams  of  finely  ground  meat/  as  free  as 
possible  from  fat  and  gristle,  are  thoroughly  mixed  with  800 
cc.  of  water  in  a  large  dish  and  heated  on  a  water-bath.  Dip 
a  thermometer  into  the  mixture.  It  should  show  a  tempera- 
ture of  50°  to  55°.     After  20  minutes  to  half  an  hour,  filter 

^  Beef  or  rabbit's  flesh  (the  latter  is  very  rich  in  creatine  and  especially 
suitable),  or  also  dog's  flesh.  Horse-flesh  is  not  suitable,  as  the  extracts 
after  the  precipitation  with  basic  lead  acetate  do  not  give  a  clear  filtrate, 
presumably  on  account  of  the  greater  amount  of  glycogen  in  horse-flesh. 

20 


EXAMINATION  OF  MUSCULAR    TISSUE.  21 

through  musUn  and  press  out  the  residue  completely  in  a 
press. 

Heat  the  combined  extracts  to  boiUng  in  a  thin-walled 
tinned  vessel,  stirring  constantly,  in  order  to  precipitate  the 
albumin.  The  fluid  surrounding  the  coagulum  of  albumin 
must  be  quite  clear.  If  it  is  not  clear  add  a  few  drops  of 
acetic  acid.  Filter  from  the  albumin,  which  is  colored  some- 
what red  from  the  blood-pigment  mixed  with  it,  and  let  cool 
completely.  The  extract,  entirely  free  from  albumin,  is 
cautiously  treated  with  basic  lead  acetate  solution  as  long  as 
a  precipitate  forms,  then  filtered,  the  filtrate  freed  from  lead  ^ 
by  means  of  hydrogen  sulphide  and  again  filtered.  Test  a 
portion  of  the  filtrate  to  see  if  it  is  free  from  lead.  When 
hydrogen  sulphide  is  passed  through  it,  no  blackening  should 
occur ;  if  it  does,  then  the  filtrate  must  be  again  treated  \\ith 
hydrogen  sulphide.  The  filtrate  is  now  evaporated,  on  the 
water-bath,  to  the  consistency  of  a  thin  sirup,  and  this  is 
allowed  to  stand  for  some  days  in  a  cool  place.  The  creatine 
crystalUzes  out.  It  is  separated  from  the  mother-liquor  by 
filtering,  if  it  separates  in  large  crystals,  or,  if  it  crystalhzes 
in  small  crystals,  by  pouring  the  whole  mass  on  a  porous 
plate.     It  is  then  recrystallized  from  a  Httle  hot  water. 

Creatine,  C4HgN302+H20,  forms  transparent,  colorless, 
hard,  rhombic  prisms,  which  easily  lose  their  water  of  crys- 
taUization.  It  dissolves  in  74  parts  of  cold  water,  more 
readily  in  hot,  very  sUghtly  in  alcohol,  and  not  at  all  in  ether. 
The  solutions  react  neutral.  Creatine  has  no  characteristic 
reactions.  For  its  recognition  we  make  use  of  either  its  con- 
duct when  heated  cautiously  or  its  conversion  into  creatinine. 

1.  A  small  portion  of  the  creatine  is  cautiously  heated 

*  Instead  of  this  we  may  also,  in  order  to  save  time,  precipitate  the 
greater  part  of  the  lead  by  the  cautious  addition  of  sulphuric  acid  and 
then  separate  the  remainder  by  means  of  hydrogen  sulphide;  however, 
the  lead  sulphate  passes  readily  through  the  filter. 


22     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

on  a  crucible  cover  or  a  piece  of  platinum  foil  over  a  very- 
small  flame.  It  first  loses  its  water  of  crystallization  and 
becomes  whit;e  like  porcelain,  then  it  turns  brown,  giving  off 
a  characteristic  odor,  carbonizes,  and  finally  burns  without 
leaving  any  residue — if  pure. 

2.  Conversion  into  Creatinine.  Heat  the  remainder  of 
the  creatine  for  half  an  hour  with  10  cc.  of  dilute  sulphuric 
acid  (20  per  cent.)  on  the  water-bath,  adding  water  to  re- 
place that  lost  by  evaporation.  To  get  rid  of  the  sulphuric 
acid  grind  the  solution,  after  the  addition  of  water,  in  a  mor- 
tar with  bariiun  carbonate  until  the  mixture  no  longer  reacts 
acid,  filter,  and  evaporate  the  filtrate  on  the  water-bath  to  a 
few  cubic  centimeters. 

(a)  Creatinine  Zinc  Chloride,  (C4H7N30)2ZnCl2.  In  a 
watch-glass  add  to  a  few  drops  of  the  solution  thus  obtained  a 
drop  of  an  alcoholic  solution  of  zinc  chloride.  A  pulverulent 
or  microcrystalline  precipitate  of  creatinine  zinc  chloride 
soon  separates.    Examine  under  the  microscope. 

(6)  Weyl's  Reaction.  Mix  the  greater  part  of  the  solu- 
tion with  sodium  nitroprusside  solution  (freshly  prepared  by 
dissolving  a  few  crystals  in  a  little  water)  until  the  solution 
has  a  yellow  color,  then  add  a  few  drops  of  caustic  soda  solu- 
tion. The  fluid  turns  deep  red  to  ruby-red,  the  color  soon 
fades  and  becomes  straw-yellow.  If  the  solution  is  now 
acidified  with  glacial  acetic  acid  (about  one-fourth  the  vol- 
imie)  and  heated  to  boiling  or  allowed  to  stand  for  some  time 
it  turns  green,  and  deposits  on  longer  standing  a  precipitate 
of  Prussian  blue. 

The  mother-hquor  from  the  creatine  contains  the  xan- 
thine bases  or  alloxuric  bases  (earher  also  called  xanthine 
bodies),  which  may  be  obtained  in  the  form  of  the  silver 
compounds  by  adding  ammonia  to  alkahne  reaction,  then 
filtering  and  adding  an  ammoniacal  silver  nitrate  solution 
(see  below). 


EXAMINATION  OF  MUSCULAR  TISSUE.  23 

The  residue  of  meat  remaining  after  pressing  is  broken  up, 
heated  to  boiling  with  water  in  a  tin  dish,  and  the  water 
together  with  the  fat  floating  on  it  is  poured  off.  This  opera- 
tion is  repeated  once  more  and  then  the  mass  is  filtered 
through  musHn.  The  residue  may  be  used  for  digestion 
experiments. 

II.   DETECTION  OF  THE   PRINCIPAL  PROTEIDS  IN  MEAT. 

Meat  extracted  with  cold  water. 


Filtrate  contains  soluble  Residue   may  be   used  for  the 

proteids.  preparation  of  myosin. 

Mix  100  g.  of  finely  ground  meat  with  300  cc.  of  water, 
s;tir  thoroughly,  let  stand  one  to  two  hours,  pour  the  mixture 
through  a  musUn  filter  and  squeeze  out  the  residue  with,  the 
hand.  The  filtrate  is  colored  red  (from  the  haemoglobin)  and 
is  usually  somewhat  cloudy  on  account  of  the  fat  and  small 
particles  of  muscle.  In  order  to  make  it  clear  it  is  filtered 
through  paper. 

1.   Filtrate. 

(a)  Testing  the  Reaction.  Tested  with  sensitive  Utmus 
paper  the  filtrate  reacts  acid  in  consequence  of  its  containing 
primary  potassium  phosphate  (KH2PO4).  The  acid  reaction 
becomes  stronger  on  keeping  the  meat  on  account  of  the 
formation  of  lactic  and  other  acids. 

(6)  A  portion  of  the  filtrate  is  slowly  heated  in  a  test- 
tube  in  which  a  thermometer  is  placed.  For  this  purpose 
put  the  test-tube  in  a  beaker  half  filled  with  water  and  heat 
the  beaker  on  the  wire  gauze.  By  frequently  stirring  the 
water  with  a  glass  rod,  on  the  under  end  of  which  is  a  piece 
of  rubber  tubing,  the  even  distribution  of  the  temperature 
may  be  accomplished.  Even  at  a  slight  elevation  of  tem- 
perature, usually  at  55°  to  56°,  coagulation  occurs,  the  fil- 


24     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

trate  from  the  coagulum  again  coagulates  at  about  65°,  and 
the  filtrate  from  this  at  about  75°. 

2,  Residue.  Preparation  of  Myosin.  The  meat  residue 
is  again  extracted  in  the  same  manner  with  water,  the  water 
poured  off,  and  then  the  residue  stirred  into  a  thin  paste  with 
a  15  per  cent,  solution  of  ammonium  chloride,  and  after 
twenty-four  hours  filtered.     The  solution  contains  myosin. 

(a)  A  part  of  the  solution  is  poured  into  a  test-tube  two- 
thirds  full  of  water.  Separation  of  myosin  in  a  swollen  con- 
dition. 

(&)  A  part  of  the  solution  is  poured  upon  a  piece  of  salt 
in  a  small  beaker.  The  surface  of  the  salt  becomes  covered 
with  the  precipitated  myosin.  Instead  of  this  we  may  also 
cause  the  separation  of  the  myosin  by  introducing  finely  pul- 
verized salt  into  the  solution  and  stirring. 

(c)  A  portion  of  the  solution  is  heated  to  boihng  and  fil- 
tered. The  filtrate  contains  calcium  salts,  as  may  be  shown 
by  the  addition  of  ammonium  oxalate. 

Myosin  is  therefore  characterized  by  its  insolubihty  in 
water  and  strong  solutions  of  salts,  solubility  in  salt  solutions 
of  medium  concentration,  and  by  the  fact  that  it  contains 
calcium,  which  it  gives  up  on  coagulation. 

III.  Preparation  of  the  Xanthine  Bases,  Alloxuric 

BASES. 

Method  A. 

Dissolve  50  g.  of  meat  extract  in  500  cc.  of  water  in  a 
flask  and,  after  the  addition  of  75  to  100  cc.  of  nitric  acid 
(1.2  sp.  gr.)  to  destroy  substances  wjiich  hinder  the  precipi- 
tation of  the  xanthine  bases  by  silver  nitrate  heat  on  the 
sand-bath  until  the  solution  has  cleared  up  which  will  require 
about  three-quarters  of  an  hour.  After  cooling  make  strongly 
alkaline  with  ammonia,  filter  from  the  phosphates  which 


EXAMINATION  OF  MUSCULAR   TISSUE.  25 

separate,  and  add  an  ammoniacal  solution  of  2.5  g.  of  silver 
nitrate  in  about  100  cc.  of  water.  The  precipitate,  which 
consists  for  the  most  part  of  hypoxanthine  silver  besides  a 
little  xanthine  silver,  is  then  collected  on  a  filter  and  washed 
a  few  times  with  water. 

Separation  of  Hypoxanthine  and  Xanthine. 

The  separation  of  these  two  xanthine  bases  is  accomplished 
by  converting  them  into  the  silver  nitrate  compounds.  These 
compounds  conduct  themselves  differently  towards  nitric 
acid.  The  hypoxanthine  silver  nitrate  compound  is  very 
difficultly  solub'e  in  nitric  acid;  the  xanthine  silver  nitrate 
compound  is  far  more  readily  soluble.  The  following  is  the 
best  method  of  procedure: 

Put  the  still  moist  precipitate  into  a  flask  and  pour  over 
it  a  mixture  of  100  cc.  of  nitric  acid  and  100  cc.  of  water, 
add  1  g.  of  urea,  heat  just  to  boihng,  and  let  cool.  The 
hypoxanthine  silver  contained  in  the  precipitate  is  converted 
into  hypoxanthine  silver  nitrate,  which  remains  partly  un- 
dissolved and  partly  goes  into  solution,  but  separates  out  of 
the  solution  again  on  cooling.  The  addition  of  the  urea  is 
to  prevent  the  formation  of  nitrous  acid,  which  might  decom- 
pose the  xanthine  bases.  Filter  the  hypoxanthine  silver 
nitrate  off  after  a  few  hours  and  wash  unti!  the  wash-water 
no  longer  reacts  strongly  acid.  Let  the  filtrate  (without  the 
wash-water)  stand  till  next  day  and  filter  (without  working 
up  the  precipitate,  which  is  a  mixture  of  hypoxanthine  silver 
nitrate  and  xanthine  silver  nitrate).  The  filtrate  is  used  to 
show  the  presence  of  xanthine.  The  hypoxanthine  silver 
nitrate  is  examined  under  the  microscope  (fine  needles  fre- 
quently grouped  in  the  form  of  stars). 


26     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

Conversion  of  the  Hypoxanthine  Silver  Nitrate  into 
Hypoxanthine. 

(a)  By  Means  of  Hydrochloric  Acid.  Pierce  the  filter  and 
wash  the  precipitate  into  a  flask.  Add  a  few  cubic  centi- 
meters of  hydrochloric  acid,  shake  thoroughly  and  continu- 
ously, and  finally  warm  gently.  The  hydrochloric  acid  de- 
composes the  silver  compound  with  the  separation  of  silver 


Fig.  1. — Hypoxanthine  Silver  Nitrate, 

chloride.  By  thoroughly  shaking  and  gently  warming  this 
settles  well.  The  completion  of  the  decomposition  is  con- 
trolled by  a  microscopical  examination.  Heating  too  strongly 
is  to  be  avoided,  aB  otherwise  the  aqua  regia  formed  may 
destroy  the  hypoxanthine.^  When  the  decomposition  is  com- 
plete, filter,  make  the  filtrate  alkaline  with  ammonia,  and 
evaporate  to  dryness  on  the  water-bath.  Treat  the  residue 
with  a  small  quantity  of  water,  which  dissolves  the  ammo- 
nium chloride  and  nitrate  formed,'  but  leaves  the  hypoxan- 
thine undissolved. 

^  In  order  to  avoid  this  it  is  advisable  first  to  convert  the  hypoxan- 
thine silver  nitrate  into  the  hypoxanthine  silver  compound  (see  below). 


EXAMINATION  OF  MUSCULAR   TISSUE.  27 

(b)  By  Means  of  Hydrogen  Sulphide.  Put  the  hypoxanthine  silver 
nitrate  into  a  flask  with  some  water  and  pass  in  hydrogen  sulphide, 
shaking  frequenth',  untH  the  precipitate  appears  perfectly  black  and 
no  white  particles  are  to  be  seen.  Filter:  the  filtrate  contains  the 
hj'poxantliine  nitrate.  Concentrate  somewhat  on  the  water-bath  in 
order  to  drive  off  the  hydrogen  sulphide,  make  faintly  alkaline  with 
ammonia,  and  proceed  as  above.  The  disadvantage  of  this  method 
is  that  the  complete  decomposition  with  hydrogen  sulpliide  is  difficult 
to  accomplish  and  that  the  h3'poxanthine  may  contain  some  sulphur. 

The  decomposition  may  be  accomplished  more  readily  if  the  hypo- 
xanthine sUver  nitrate  is  first  converted  into  the  silver  compound  of 
h}iDOxanthine  by  digesting  it  for  some  time  with  water,  ammonia,  and 
2  g.  of  silver  nitrate.  Filter  and  wash  the  precipitate,  suspend  it  in 
water,  warm,  and  add  ammonium  sulphide,  drop  by  drop.  The  silver 
sulphide  settles  on  warming;  the  filtrate  on  evaporation  yields  hypo- 
xanthine (Scliindler  '). 

For  the  reactions  the  hj^DOxanthine  obtained  in  any  one  of  the 
above  ways  is  sufficiently  pure.  In  case  it  is  markedly  yellow  it  maj' 
be  purified  in  the  following  manner:  Dissolve  the  hypoxanthine  in 
water  by  adding  hydrochloric  acid,  add  a  few  drops  of  ferrous  sulphate, 
heat  the  solution  and  make  it  alkaline  with  sodium  hydroxide,  filter, 
concentrate  somewhat,  and  precipitate  the  hypoxanthine  by  acidify- 
ing faintly  with  acetic  acid  or  by  neutralizing  exactly  with  hydro- 
chloric acid.  Iron,  in  the  form  of  ferrous  hydroxide,  often  passes  into 
the  filtrate  on  filtering  the  alkaline  solution.  It  is  then  necessary  to 
let  the  filtrate  stand  for  some  time,  shaking  it  frequently  until  the 
iron  has  completely  separated  as  ferric  hydroxide. 

Method  B. 

In  the  method  A,  owing  to  the  action  of  the  nitric  acid  on 
the  xanthine  bases,  so-called  nitro  compounds  may  easily  be 
formed  and  contaminate  the  hypoxanthine.  This  may  be 
avoided  by  getting  rid  of  the  substances,  which  interfere 
with  precipitation  of  the  xanthine  bases,  not  by  oxidation 
with  nitric  acid,  but  by  precipitation.  Dissolve  50  g.  of  meat 
extract  in  500  cc.  of  water,  add  basic  lead  acetate  to  the 

'  Zcitschr.  f.  physiol.  Chem.,  13,  433. 


2S     PHYSIOLOGICAL  AXD  PATHOLOGICAL   CHEMISTRY. 

solution  as  long  as  a  precipitate  is  formed,  filter,  and  remove 
the  excess  of  lead  from  the  filtrate  with  hydrogen  sulphide. 
Boil  the  solution  or  concentrate  it  on  the  water-bath,  in 
order  to  get  rid  of  the  excess  of  hych'ogen  sulphide,  make 
strongly  alkahne  with  ammonia,  and  precipitate  with  an 
ammoniacal  solution  of  silver  nitrate,  etc.,  etc. 

The  hypoxanthine  (or  sarcine),  C5HJN4O,  is  very  diffi- 
cultly soluble  in  cold  water  (in  1400  parts  at  19°),  somewhat 
more  soluble  in  hot  water  (70  parts).  It  dissolves  readily  in 
mineral  acids,  forming  weU-crystalhzed  salts,  and  in  alkalies, 
even  in  ammonia  (distinction  from  guanine,  which  is  but 
very  shghtly  soluble  in  ammonia).  It  gives  the  so-called 
xanthine  reaction,  but  less  markedly  than  any  of  the  other 
xanthine  bases. ^ 

Reactions  of  Hypoxanthine. 

1.  Pom'  upon  a  small  portion  of  the  substance  on  a  por- 
celain crucible  cover  a  few  drops  of  strong  or  finning  nitric 
acid  and  evaporate  cautiously  to  dryness  over  a  small  flame. 
A  lemon-yellow  residue  results,  wliich  takes  on  an  orange 
color  when  moistened,  after  coohng,  ^^ith  caustic  soda  solu- 
tion. If  a  drop  of  water  be  then  added,  a  yellow  solution 
results,  and  tliis  when  evaporated  again  leaves  an  orange 
residue  (distinction  from  the  mm-exide  reaction  for  mic  acid). 

2.  Pour  upon  a  small  portion  of  the  substance  in  a  dish  a 
little  pme  nitric  acid  of  the  specific  gra^^ity  1.2,  and  evaporate 
on  the  water-bath  to  drjmess.  The  residue  is  scarcely  per- 
ceptibly colored.  On  the  addition  of  caustic  soda  it  becomes 
pale  yellow  (distinction  from  xanthine  and  guanine,  which 
under  these  conditions  give  the  xanthine  reaction).  Hj-po- 
xanthine  is  further  distinguished  by  its  solubility  in  ammonia 

^  According  to  Hoppe-Seyler,  Thierfelder,  7th  edition,  page  147,  typo- 
xanthine  does  not  give  the  xanthine  reaction, — O. 


EXAMIXATIOX  OF  MUSCULAR    TISSUE.  29 

and  the  insolubility  of  its  compoimd  ^^-ith  silver  nitrate  in 
nitric  acid,  and  also  by  the  crystal  form  of  this  compound. 

Detection  of  Xanthine. 

The  filtrate  from  hypoxanthine  silver  nitrate  contains 
xanthine,  as  previously  stated,  but  only  in  small  quantity. 
Make  it  alkahne  with  ammonia  (or,  in  order  to  save  ammonia, 
neutrahze  the  greater  part  of  the  acid  xsith  soda  or  hme  and 
then  make  alkahne  with  anmionia).  Xanthine  silver  pre- 
cipitates in  brown  or  reddish  flakes.  These  are  filtered  off, 
washed,  saspended  in  water,  some  drops  of  ammonia  added, 
heated,  treated  with  a  few  drops  of  ammonimn  sulpMde, 
shaken  thoroughly,  filtered  from  the  silver  sulphide,  and 
evaporated  (or  the  precipitate  may  also  be  decomposed  with 
hydrochloric  acid  and  xanthine  hydrochloride  obtained  on 
evaporation).  Very  frecj[uently  the  silver  sulphide  passes 
through  the  filter;  we  then  evaporate  to  dryness  and  extract 
the  residue  with  boiUng  water.  The  xanthine  thus  obtained 
is  usually  not  quite  pure  and  the  cpantity  is  very  small.  It 
suffices,  however,  for  the  xanthine  test  as  well  as  for  the  so- 
called  Weidel's  reaction. 

Xanthine,  CjH^X^O,,  occurring  very  rarely  in  the  form  of 
stone  in  the  bladder,  results,  like  the  hypoxantliine,  from  the 
decomposition  of  the  nucleins  (A.  Ko5sel).  In  cold  water 
it  is  practically  insoluble  (14,151  parts  at  16°).  In  alcohol 
and  ether  it  is  insoluble.  In  hot  water  it  is  very  difficultly 
soluble,  but  it  is  soluble  in  caustic  soda  and  ammonia  as  well 
as  in  acids,  forming  salts. 

1.  Xanthine  Test.  Dissolve  the  residue  or  half  of  it  in 
nitric  acid  and  evaporate  cautiously  to  dryness  on  a  crucible 
cover  over  a  small  flame.  A  lemon-yellow  residue  results, 
which  becomes  inten.sely  red  on  moistening  with  caustic  soda, 
and  on  further  heating  purplish  red.  Add  a  few  drops  of 
water  and  warm;  a  yellow  solution  results,  which  again  gives 


30     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

a  red  residue  on  evaporation  (distinction  from  the  murexide 
reaction  for  uric  acid). 

2.  The  So-called  Weidel's  Reaction.^  Dissolve  half  of  the 
xanthine  obtained  in  bromine-water,  warming  gently,  evap- 
orate the  solution  on  the  water-bath  to  dryness,  and  invert 
the  dish  over  another  which  contains  some  ammonia.  The 
residue  becomes  red.^ 

IV.  Preparation  of  sarcolatic  Acid. 

For  this  purpose  the  filtrate  from  the  silver  precipitate 
III  Method  B  may  be  used.  Evaporate  this  to  a  sirup  on  the 
water-bath.  Ammonia  escapes  and  a  portion  of  the  silver 
separates  in  reduced  form.  Extract  the  residue  with  alcohol, 
filter  the  alcohoHc  extract,  evaporate  on  the  water-bath,  dis- 
solve in  75  cc.  of  water  to  which  25  cc.  of  dilute  sulphuric 
acid  have  been  added,  and  extract  by  shaking  in  a  separat- 
ing-funnel,  three  or  four  times  at  least,  with  one  and  a  half 
volumes  of  ether  to  which  a  httle  alcohol  has  been  added. 
Separate  the  ether,  filter  through  a  dry  filter,  and  distil  off 
the  ether.  It  is  advantageous  to  distil  the  first  extract  at 
once  and  use  the  distilled  ether  (adding  some  fresh  ether) 
for  the  second  extraction. 

Use  a  small  portion  of  the  residue  resulting  for  the  Uffel- 
mann's  reaction  for  lactic  acid  (see  chapter  on  Digestion), 
Dissolve  the  remainder  in  water,  boil  with  freshly  precipi- 

^  The  best  way  to  perform  the  test  according  to  E  Fischer  (Ber.  30, 
2236  (1897)  is  to  boil  a  small  quantity  of  the  finely  powdered  xanthine 
with  some  freshly  prepared  chlorine-water  or  with  some  hydrochloric 
acid  and  a  little  potassium  chlorate,  then  cautiously  evaporate  the  fluid 
to  dryness  on  platinum  foil  and  moisten  the  residue  with  ammonia  (forma- 
tion of  murexide). — O. 

^  If  we  use,  in  addition  to  the  bromine-water  (originally  chlorine-water 
was  prescribed),  a  trace  of  nitric  acid,  the  reaction  is  far  more  beauti- 
ful, but  under  these  conditions  other  xanthine  bases  also  give  the  test. 
This  form  of  the  reaction  is  therefore  to  be  discarded  as  liable  to  lead  to 


EXAMIXATION  OF  MUSCULAR   TISSUE.  31 

tated  basic  zinc  carbonate,  filter,  evaporate  to  crystallization 
(examine  microscopically),  and  when  this  has  begun  add 
alcohol.  The  zinc  salt  resulting  is  filtered  off  and  freed 
from  the  mother-liquor  c'inging  to  it  by  pressing  between 
filter-paper.     If  it  is  quite  free  from  sulphuric  acid  ^  (after 


Fig.  2. — Zinc  Sarcolactate. 

purification  by  recrystallization)  make  a  determination  of 
the  amount  of  the  water  of  crystallization.  For  this  purpose 
weigh  off  on  a  watch-glass  exactly  0.3  to  0.5  g.  of  the  air-dried 
zinc  salt,  and  heat  for  some  time  in  an  air-bath  at  115°  until 
the  weight  is  constant.  Zinc  sarcolactate  crystallizes  with 
two  molecules  of  water,  (C3H503)2Zn  +  2H20  (12.89  per  cent. 
HjO).     The  loss  in  weight  must  therefore  be  12.89  per  cent. 

Digestion  with  water  containing  chloroform  is  a  very  convenient 
method  for  the  preparation  of  the  xanthine  bases.  Five  hundred 
grams  of  finely  divided  meat  are  placed  in  a  glass-stoppered  bottle,  or 
divided  between  two  bottles,  with  5  liters  of  chloroform-water  (5  cc. 
of  chloroform  to  1  liter;  in  this  case  ordinary  water  may  be  used), 
then  a  few  drops  more  of  chloroform  are  added,  and  the  mixture  is  well 
shaken. 


'  The  presence  of  the  sulphuric  acid  may  be  avoided  if  phosphoric  acid 
in.stf;ad  of  sulphuric  acid  is  used  to  acidifj'. 


32     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

Digest  for  two  to  three  days  at  40°,  shaking  repeatedly  and  thor- 
oughly, then  filter  and  drain  the  residue  completely  by  pressure. 
Heat  the  extract  in  order  to  coagulate  the  albumin,  evaporate  the 
filtrate  to  about  500  cc,  make  strongly  alkaline  with  ammonia,  and 
precipitate  with  ammoniacal  silver  solution,  etc.  By  means  of  the 
digestion  the  nuclein  is  decomposed  and  the  substances  which  prevent 
the  preciijitation  of  the  hypoxanthine  silver  are  removed. 

V.  Detection  of  Organically  Combined  Phosphorus  in 
Meat  Extract. 

According  to  Siegfried,  meat  extract  free  from  albumin 
contains  an  organic  substance  (nucleon)  in  which  phosphorus 
is  present.  Carniferrin  is  the  name  given  by  Siegfried  to  the 
iron  compound  of  this  substance.  The  following  method 
may  be  used  for  the  preparation  of  this  compound :  Dissolve 
10  g.  of  meat  extract  in  200  cc.  of  water,  make  faintly  alka- 
line with  ammonia,  add  calcium  chloride  solution  as  long  as 
a  precipitate  forms  (the  reaction  must  remain  neutral  during 
the  precipitation  and  later  a  httle  more  ammonia  is  to  be 
added),  and  filter.  To  the  filtrate  add  15  cc.  of  a  3  per  cent, 
ferric  chloride  solution,  heat  to  boihng  and,  if  necessary, 
neutrahze  exactly  vdth  ammonia.  The  precipitate  is  fil- 
tered off  and  washed.  It  dissolves  in  dilute  sodium  car- 
bonate solution,  forming  a  more  or  less  clear  solution.  The 
remainder  of  the  carniferrin  is  ground  with  alcohol,  filtered, 
and  then  treated  with  a  small  quantity  of  ether.  The  pres- 
ence of  the  phosphorus  may  be  shown  by  fusing  with  the 
oxidizing  mixture,  dissolving  the  fused  mass  in  water,  filter- 
ing from  the  iron  oxide,  and  testing  for  phosphoric  acid  in 
the  filtrate. 


CHAPTER  III. 

GASTRIC  DIGESTION. 

I.  Detection  of  Hydrochloric  Acid  in  the  Gastric  Juice: 

(a)  with  methyl  violet;    (b)  -with  tropseolin;    (c)  with  Giins- 
burg's  reagent. 
II.  Detection  of  Lactic  Acid  (and  Hydrocliloric  Acid) : 

(a)  by  Uffelmann's  method;   (6)  after  extraction  with  ether. 

III.  Detection  of  Pepsin  in  Gastric  Juice  or  Vomit. 

IV.  Influence  of  the  Amount  of  Pepsin  on  the  Intensity  of  Digestion. 
V.  Influence  of  Disturbing  Substances,  Qualitative. 

VI.  Comparison  of  Different  Kinds  of  Pepsin. 
VII.  Preparation  of  the  Products  of  Digestion. 

i.  detection  of  hydrochloric  acid  in   the  gastric 

Juice. 

Solutions  required: 

1.  Six  cubic  centimeters  of  strong  hydrochloric  acid, 
specific  gravity  1.19  (about  37  per  cent.  HCl),  diluted  to 
one  liter:  solution  A.  This  solution  contains  0.27  per 
cent,  of  HCl. 

2.  One  hundred  cubic  centimeters  of  solution  A  diluted 
to  500  cc:   solution  B. 

3.  Eight-tenths  of  a  gram  of  lactic  acid  dissolved  in  100 
cc.  of  water. 

4.  Two  grams  of  commercial  peptone  dissolved  in  100  cc. 
of  water. 

33 


34     PHYSIOLOGICAL  AXD  PATHOLOGICAL   CHEMISTRY. 

(a)  Reactions  with  Methyl  Violet  (0.5  :  1000)  or  Gentian 

Violet. 

1.  A  small  portion  of  the  hj'drochloric  acid  solution  A  in 
a  test-tube  is  treated  T\i.th  a  few  drops  of  methyl  violet  solu- 
tion: steel-blue  color.  Make  the  same  test  Tiith  water: 
violet  color.  With  the  lactic  acid  solution:  Aiolet  with  a 
hght-bluish  tint. 

2.  Influence  of  Dilution.  Repeat  experiment  I  T\i.th  the- 
hydrochloric  acid  solution  B. 

3.  Influence  of  the  Presence  of  Albumoses  and  Peptone. 
Dilute  some  of  the  hydrochloric  acid  solution  A  ^ith  an 
equal  A'olume  of  water,  and  another  portion  \\ith  an  ec^ual 
volume  of  the  peptone  solution,  then  add  a  few  drops  of  the 
methyl  \dolet  solution  to  both  and  compare  the  colors.  We 
may  also  perform  the  experiment  by  taking  some  of  the  steel- 
blue  solution,  produced  by  the  action  of  the  hydrochloric: 
acid  (A)  on  the  methyl  violet  solution,  di^ichng  it  into  twa 
parts,  and  adcUng  the  peptone  solution  to  one  part  and  water 
to  the  other  part. 

4.  Influence  of  the  Peptone  in  very  dilute  Hydrochloric 
Acid.  Repeat  experiment  3  \vith  the  hydrochloric  acid 
solution  B.  From  the  results  in  3  and  4  it  will  be  seen  that 
the  methyl  ^iolet  reaction  cannot  be  used  in  the  presence  of 
considerable  quantities  of  albumoses  and  peptone. 

5.  "(a)  To  about  30  cc.  of  the  lactic  acid  solution  add 
some  of  the  methyl  violet  solution  and  divide  the  mixture 
into  three  parts.  To  the  first.  A',  add  an  equal  volume  of 
water;  to  the  second,  B',  the  same  volume  of  water  and  then 
satm-ate  the  mixture  "uith  salt  (XaCl);  to  the  third,  C,  the 
same  volume  of  a  3  per  cent,  salt  solution.  A'  does  not 
change  its  color,  it  only  becomes  somewhat  clearer;  C  con- 
ducts itself  in  the  same  waj'-;  B',  howe^ver,  becomes  perceptibly 
steel-blue.     Conclusion:   sodium  chloride  in  the  presence  of 


GASTRIC  DIGESTION.  35 

lactic  acid  interferes  with  the  reaction  only  in  quite  concen- 
trated solution,  owing  to  the  hydrochloric  acid  set  free. 

(6)  Repeat  experiment  5  (a),  using  a  solution  (0.25  g.  to 
1000  cc.)  of  tropseoUn  00/  instead  of  the  methyl  violet. 

The  reactions  may  be  made  sharper  by  cautious  evapo- 
ration of  the  mixtures  (about  30  drops)  in  porcelain  dishes. 

(c)  Reactions  with  Gunzburg's  Reagent.  1  g.  vanilUn, 
2  g.  phloroglucin,  100  cc.  alcohol.^ 

1.  Add  a  drop  of  Gunzburg's  reagent  to  a  few  drops  of 
the  hydrochloric  acid  solution  A,  evaporate  to  dryness  in  a 
small  porcelain  dish  over  a  free  flame.  Avoid  heating  too 
strongly  by  moving  the  contents  of  the  dish  from  side  to 
side  and  blowing  upon  it  during  the  heating:  purple-red 
residue. 

Repeat  the  experiment  with  the  mixtures  2,  3,  and  4. 
The  peptone  interferes  with  the  Gimzburg  reaction  less  than 
with  the  previous  ones.  Lactic  acid  does  not  give  the 
reaction.  ^ 

II.  DETECTION  OF  LACTIC  ACID. 
(a)  With  Uffelmann's  Reagent.     To  prepare  this  reagent 
add  to  10  cc.  of  a  2  per  cent,  phenol  solution  a  few  drops  of 
ferric  chloride:    amethyst-blue  fluid. 

'  Only  the  tropaeolin  with  this  trade  name  is  to  be  used. 

^  Gunzburg's  reagent  cannot  be  kept  long  without  undergoing  change, 
and  it  works  best  when  it  is  freshly  made.  The  quantities  do  not  make 
much  difference,  so  that  we  can  prepare  the  reagent  by  dissolving  as 
much  phloroglucin  as  may  be  held  on  the  point  of  a  small  knife-blade 
and  the  same  quantity  of  vanillin  in  a  few  cubic  centimeters  of  alcohol  in 
a  test-tube. 

*  An  alcoholic  solution  (0.2  per  cent.)  of  dimethylamidoazobenzene  is 
more  sensitive  towards  free  hydrochloric  acid  than  Gunzburg's  reagent. 
The  yellow  solution  is  changed  to  red  in  the  presence  of  free  hydrochloric 
acid.  Dilute;  solutions  of  the  organic  acids  do  not  give  the  reaction. 
Filt^ir-paper  dipped  into  the  above  solution  of  dimethylamidoazobenzene 
and  allowed  to  dry  may  also  be  used  v(;ry  advantageously  to  detect  small 
quantities  of  free  hydrochloric  acid  in  the  gastric  contents.  See  J.  Fried- 
en  wald,  Medical  Record,  April  6,  189.5. — O. 


36     PHYSIOLOGICAL  A.VD  PATHOLOGICAL  CHEMISTRY. 

1.  Add  a  few  drops  of  the  reagent  to  some  of  the  stronger 
hydrochloric  acid  solution  A:    decolorization. 

2.  Eepeat  with  the  lactic  acid  solution:  lemon-yellow 
color. 

3.  Repeat  with  a  mixture  of  equal  parts  of  the  hydro- 
chloric acid  solution  A  and  lactic  acid:  lemon-yeUow  color, 
but  fainter  than  in  2.  Conclusion:  lactic  acid  in  the  pres- 
ence of  hydrochloric  acid  may  be  detected  by  Uffehnann's 
reagent,  but  not  hydrochloric  acid  in  the  presence  of  lactic 
acid. 

4.  Add  to  15  cc.  of  the  lactic  acid  solution  some  of  Uffel- 
mann's  reagent  and  divide  into  three  parts,  A,  B,  and  C. 
To  A  add  its  own  volume  of  water,  to  B  the  same  voliune  of 
a  concentrated  salt  solution,  to  C  the  same  volume  of  a  3  per 
cent,  salt  solution:  only  B  is  decolorized;  the  presence  of 
sodium  chloride,  therefore  does  not  as  a  rule  interfere  with 
the  detection  of  lactic  acid. 

(b)  After  Isolating  the  Lactic  Acid  by  Means  of  Ether.  Mix 
25  cc.  of  the  hydrochloric  acid  solution  A  with  25  cc.  of  the 
lactic  acid  solution,  shake  with  50  cc.  of  ether,  separate  the 
ether  and  shake  the  aqueous  fluid  again  with  ether.  Filter 
the  united  ether  extracts  through  a  dry  filter  and  distil  off 
the  ether.  Take  up  the  residue  in  a  httle  water  and  test  the 
solution  with  ITffelmann's  reagent.  This  method  of  isolat- 
ing the  lactic  acid  by  means  of  ether  is  used  especially  with 
the  fluids  of  the  stomach  when  their  color  prevents  or  ren- 
ders difficult  the  direct  detection  of  the  lactic  acid. 

ni-  DETECTiorr  OF  PEPsiPf  m  THE  Gastric  juice  or 

Vomit. 

Add  to  10  or  20  cc.  of  the  fluid  10  to  20  drops  of  pure 
dilute  hydrochloric  acid  (1  :  10),  then  add  some  shreds  of 
fibrin  or  a  shce  of  hard-boiled  egg-albumen,  and  keep  the 
mixture  at  40°.     The  shreds  of  fibrin  should  dissolve  even  to 


GASTRIC  DIGESTION.  37 

the  last  particle  in  a  quarter-  to  a  half-hour,  and  the  slice  of 
egg-elbumen  should  be  perceptibly  diminished  after  an  hour 
if  pepsin  is  present. 

IV.  Influence  of  the  Quantity  of  Pepsin  on  Digestion. 

Prepare  an  artificial  digestive  fluid  from  commercial  pep- 
sin and  dilute  hydrochloric  acid  (0.27  per  cent,).  If  pepsin, 
which  will  dissolve  in  water  forming  a  clear  or  almost_  clear 
solution,  is  to  be  had,  dissolve  0.5  g.  of  it  in  500  cc.  of  dilute 
hydrochloric  acid  (the  hydrochloric  acid  used  for  digestion 
experiments  is  always  a  mixture  of  6  cc.  of  strong  hydro- 
chloric acid,  sp.  gr.  1.19,  with  994  cc.  of  water,  i.e.,  6  cc. 
of  hydrochloric  acid  diluted  to  one  liter:  the  solution  desig- 
nated on  page  33  as  hydrochloric  acid  solution  A).  If 
only  the  pepsin  insoluble  in  water  is  available,  which  may 
nevertheless  be  very  active,  treat  0.5  g.  of  this  with  water, 
stir  thoroughly,  and  wash  with  water  until  the  filtrate  no 
longer  gives  the  reaction  for  milk-sugar.  After  piercing  the 
filter,  wa.sh  the  residue  into  a  flask  with  100  cc.  of  the  0.27 
per  cent,  hydrochloric  acid,  let  stand  with  frequent  agita- 
tion for  twenty-four  hours  at  the  room  temperature  or  at 
40°,  filter,  and  dilute  with  solution  A  to  one-half  of  a  liter. 

Put  into  each  of  three  test-tubes.  A,  B,  and  C,  approxi- 
mately equal  quantities  of  fibrin,^  preferably  weighed  quan- 
tities (about  1  g.).  To  A  add  10  cc.  of  the  dilute  hydrochloric 
acid  ("0.27  per  cent.),  to  B  5  cc.  of  the  same  hydrochloric 
acid  and  5  cc.  of  the  pepsin  hydrochloric  acid  solution,  to  C 
10  cc.  of  the  pepsin  hydrochloric  acid,  and  place  the  tubes  in 
a  water-bath  at  40°.  The  fibrin  in  Aswels,  but  does  not  dis- 
solve; in  B  and  C  it  dissolves,  and  more  quickly  in  C  than  in 
h.     If  the  pepsin  used  is  very  active  it  may  happen  that  no 

'  Instead  of  fresh  fibrin  we  may  also  use  here  the  fibrin  which  has  been 
kept  in  glycerin,  after  it  has  been  freed  from  the  glycerin  clinging  to  it  by 
washing  thoroughly  with  water. 


38     PHYSIOLOGICAL  AND  PATHOLOGICAL   CHEMISTRY. 

difference  between  B  and  C  is  perceptible;  then  more  dilute 
solutions  are  to  be  used. 

The  experiment  may  also  be  performed  by  placing  the  fibrin  and 
the  dilute  hydrochloric  acid  (0.27  per  cent.)  in  all  the  tubes,  adding 
nothing  to  A,  to  B  2  drops  of  a  glycerin  extract  of  a  pig's  stomach, 
and  to  C  4  drops  of  the  same  extract. .  According  to  Griitzner  the 
difference  may  be  more  readUy  perceived  if  we  use  fibrin  colored  with 
carmine.  In  order  to  color  the  fibrin  let  it  lie  for  twenty-four  hours 
in  a  1  per  cent,  solution  of  carmine  (rendered  as  nearly  neutral  as 
possible  by  evaporating  the  ammonia)  and  then  wash  the  fibrin  with 
water, 

V.  Interference  of  Certain  Substances  with 
Digestion. 

Dissolve  2.5  g.  of  gum  arable,  by  shaking  for  some  me 
without  heating,  in  10  cc.  of  pepsin  hydrochloric  acid  (A'). 
In  an  equal  amount  of  the  pepsin  hydrochloric  acid  dissolve 
5  g.  of  cane  sugar  (B').  A  third  portion  is  prepared  without 
any  addition  (C).  In  each  of  these  tubes  put  1  g.  of  fresh 
fibrin  (or  of  the  fibrin  preserved  in  glycerin,  after  it  has  been 
well  washed  and  drained),  and  digest  at  40°.  In  C  the  fibrin 
dissolves  quickly,  slowly  in  B',  and  still  more  slowly  in  A^ 
Many  other  indifferent  substances,  which  have  no  affinity  for 
hydrochloric  acid,  may  also  retard  digestion. 

VI.  Comparison  of  the  different  kinds  of  Commercial 

Pepsin. 

The  method  is  somewhat  different  according  as  the  pepsin 
is  soluble  in  water  or  not.  In  the  first  case  1  g.  is  dissolved 
directly  in  500  cc.  of  0.27  per  cent,  hydrochloric  acid.  In 
the  second  case  1  g.  is  freed  from  milk-sugar  as  directed 
under  IV,  page  37,  and  then  washed  into  a  flask  with  500  cc. 
of  0.27  per  cent,  hydrochloric  acid  (or  it  may  also  be  treated 
directly,  without  any  previous  washing,  with  the  0.27  per 
cent,  hydrochloric  acid),  digested  in  this  for  twenty-four  hours 
at  room  temperature  or  at  40°,  and  then  filtered  through  a 


GASTRIC  DIGESTION.  39 

dry  filter.  In  a  number  of  test-tubes  digest  equal  weights  of 
well-drained  fibrin  with  10  cc.  of  each  of  the  above  solutions 
of  pepsin  hydrochloric  acid  at  40°,  and  observe  the  difference 
in  the  tinie  of  digestion.  In  order  to  ehminate  errors  several 
tests  should  be  made  with  each  solution  of  pepsin  hydro- 
chloric acid.  We  use  first  1  g.  of  fibrin;  if  no  difference  is 
perceptible  we  make  a  series  of  experiments  with  0.5  g.  and 
2  g.  Instead  of  the  fibrin  shces  of  egg-albumen  may  be 
used.  The  more  exact  determination  can  only  be  accom- 
phshed  by  means  of  quantitative  analysis. 

VII.  Preparation  of  the  Products  of  Digestion. 

Digest  250  grams  of  fibrin  for  forty-eight  hours  at  40°  with  one  and  a 
half  liters  of  artificial  gastric  juice  and  neutralize  with  sodium  carbonate. 


Precipitate:  acid  albumin.  Filtrate:    albumoses  and  peptone. 

Saturate  with  ammonium  sul- 
phate. 


Precipitate :  albumoses,  boil  with  Filtrate :   peptone  and  ammonium 

barium  carbonate.  sulphate,  boil  with  barium  car- 

bonate. 


Hesidue :  barium     Filtrate :  albumose     Residue :  barium      Filtrate :    pep- 
sulphate,  solution.  sulphate.  tone   solution 

containing 
barium. 

The  best  material  for  digestion  is  well-drained  fresh 
blood-fibrin. 

If  only  the  coagulated  fibrin  preserved  in  cUoroform-water  is  avail- 
able, it  is  advisable  first  to  subject  it  to  the  following  treatment  before 
using:  Heat  the  fibrin  in  a  large  dish  (enamelled-iron  dish)  \\ath  w-ater 
containing  2  cc.  of  hydrocliloric  acid,  specific  gravity  1.19,  to  the  liter, 
until  it  swells  and  becomes  jelly-like.  After  cooling  the  jelly  is  ready 
for  use.  It  dissolves  almost  as  readily  as  the  fresh  fibrin.  The  swell- 
ing, however,  sometimes  remains  incomplete,  presumably  when  the 
fibrin  was  boiled  too  long  before  being  put  into  the  chloroform-water. 


40     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

If  we  desire  to  use  the  products  of  digestion  for  any  experiments  on 
animals,  it  is  essential  also  that  the  fresh  fibrin  should  be  repeatedly 
extracted,  first  with  water,  and  then  with  faintly  acidulated  water  (not 
so  much  in  order  to  cause  it  to  swell  as  for  the  purpose  of  purifying 
it) .  If  this  is  not  done,  the  products  of  digestion  may  contain  toxic 
substances  (ptomaines  or  Gautier's  leucomaines) .  For  this  extraction 
use  water  containing  1  cc.  of  hydrochloric  acid,  specific  gravity  1.19, 
to  the  liter. 

Instead  of  fibrin  we  may  use  for  the  digestion  experi- 
ments the  meat  residue^  obtained  in  the  investigation  of 
the  soluble  constituents  of  meat  (see  chapter  on  Muscular 
Tissue,  page  23).  Coagulated  egg-albumen  is  also  to  be 
recommended  as  a  very  pure  and  satisfactory  material.  The 
albumen  of  a  considerable  number  of  eggs  (about  20)  is  care- 
fully separated  from  the  yolks,  beaten  up  in  a  cylinder  with 
an  equal  volume  of  water,  exactly  neutralized  with  hydro- 
chloric acid,  filtered  from  the  precipitate  which  forms  (through 
paper  or  by  pouring  several  times  through  muslin),  the  clear 
filtrate  poured  with  constant  stirring  into  boihng  water,  and 
the  reaction  ultimately  made  very  faintly  acid  with  acetic 
acid.  Then  heat  to  active  boiling,  filter,  and  wash  the  pre- 
cipitate thoroughly  with  hot  water. 

When  fibrin  is  used,  digest  it  in  a  bottle  or  cylinder  with 
at  least  five  times  the  quantity  of  artificial  gastric  juice  for 
forty-eight  to  seventy-two  hours  at  40°.  Prepare  the  1.5  liters 
of  artificial  gastric  juice  required  as  follows:  Mix  3  g.  of  pep- 
sin with  water  in  a  porcelain  dish,  filter,  and  wash  until  the 
filtrate  no  longer  gives  the  reaction  for  milk-sugar.  Then 
dilute  9  cc.  of  hydrochloric  acid,  specific  gravity  1.19,  to  1.5 
liters  with  water.  Pierce  the  filter  containing  the  pepsin, 
wash  the  pepsin  into  a  flask  with  a  little  water,  and  add  300 
cc.  of  the  dilute  hydrochloric  acid  just  made.  Shake  thor- 
oughly, let  stand  at  room  temperature  or  at  40°  until  next 

^  In  this  case,  however,  the  products  of  digestion  will  be  contaminated 
with  gelatin-albumose  and  gelatin-peptone. 


GASTRIC  DIGESTION.  41 

day,  filter,  and  add  the  filtrate  to  the  remaining  1200  cc.  of 
the  dilute  hydrochloric  acid. 

When  the  meat  residue  from  the  400  g.  of  meat  is  used  about  2.5 
liters  of  the  artificial  gastric  juice  are  necessary;  with  the  egg-albu- 
men from  20  eggs,  2  liters.  A  glycerin  extract  of  the  stomach-lining 
(about  4  cc.  of  the  extract  to  a  liter  of  the  dilute  hydrochloric  acid, 
0.27  per  cent.)  is  also  much  used  for  digestion  experiments.  Kiihne' 
recommends  to  press  out  the  contents  of  the  rennet  glands  of  a  well- 
washed  stomach  by  scraping  with  a  spatula  and  to  digest  10  g.  of  this 
paste  with  1  liter  of  hydrochloric  acid  (containing  0.4  per  cent.  HCl) 
for  four  hours  at  40°  (Kiihne's  "normal  gastric  juice"). 

It  is  also  recommended  to  extract  the  mucous  membrane  of  the 
stomach  directly  with  hydrocliloric  acid  (0.4  per  cent.).  When  pepsin 
hydrocloloric  acid  is  used  the  principal  product  is  albumose  with  but 
little  peptone.  When  the  artificial  gastric  juice  from  the  stomach- 
lining  is  used  it  is  said  that  more  peptone  is  formed.  It  may  be 
remarked,  however,  that  the  action  of  the  hydrocliloric  acid  extract 
of  the  stomach-lining  is  not  uniform  and  is  often  very  deficient.  This 
is  due  to  the  fact  that  this  extract  still  contains  slimy,  unkno^\^l  pro- 
teids,  which  greatly  contaminate  the  products.  To  avoid  these, 
Kiihne  ^  recommends  a  purified  gastric  juice  made  as  follows:  The 
prepared  lining  from  the  fundus  of  the  pig's  stomach  is  heated  to  40° 
for  six  days  with  seven  times  the  quantity  of  dilute  hydrochloric  acid 
containing  0.5  per  cent.  HCl.  The  solution  resulting  is  then  saturated 
directly  with  ammonium  sulphate,  when  a  voluminous,  resinous  pre- 
cipitate forms.  This  is  collected,  freed  from  the  salt  solution  as  much 
as  possible  by  pressure,  washed  quickly  with  water,  and  dissolved  in 
dilute  hydrochloric  acid  (0.5  per  cent.)  which  contains  0.25  per  cent,  of 
thymol  (using  five  times  as  much  hydrocliloric  acid  as  of  the  stomach- 
lining  originally  taken).  This  solution  is  again  heated  for  some  days 
at  40°  and  then  saturated  with  ammonium  sulphate.  The  precipitate, 
"purified  pepsin,"  is  used  for  digestion  experiments  by  suspending  it 
in  the  0.27  per  cent,  hydrochloric  acid,  using  ten  times  as  much  of 
this  dilute  hydrocliloric  acid  as  of  the  stomach-lining  originally  taken. 

When  the  digestion  has  continued  for  two  to  three  days, 
the  mixture  being  repeatedly  stirred  or  shaken,  filter  the  solu- 
tion through  muslin,  heat  gently,  and  neutralize  it  in  a  large 

>  Zeitschr.  f.  Biol.  19,  184.  ^  jbij.  22,  42G,  428. 


42     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

dish  with  sodiiun  carbonate  solution,  and  filter  from  the  pre- 
cipitate formed.  Show  that  the  precipitate  consists  essen- 
tially of  acid  albumin  (solubiUty  in  dilute  alkalies,  precipita- 
tion by  mineral  acids).  The  filtrate  is  evaporated,  at  first  by 
heating  it  to  boiling  over  a  free  flame.  Some  proteid,  appar- 
ently globuhn,  always  precipitates  at  this  point.  This  is 
filtered  off  before  the  solution  has  been  much  concentrated. 
During  the  evaporation  the  reaction  must  be  kept  as  nearly 
neutral  as  possible  (by  means  of  sodiimi  carbonate  or  dilute 
hydrochloric  acid).  Evaporate  on  the  water-bath  to  about 
200  cc.^  It  is  now  necessary  to  separate  the  albumoses  from 
the  peptone.  This  may  be  done  by  saturating  the  solution, 
acidified  with  acetic  acid,  with  sodium  chloride,  or  by  com- 
pletely saturating  it  with  ammoniimi  sulphate.  The  albu- 
moses are  precipitated,  while  peptone  remains  in  solution. 
The  advantage  of  ammonium  sulphate  over  acetic  acid  and 
salt  hes  in  the  more  complete  precipitation  of  the  albumoses ; 
however,  even  when  ammonium  sulphate  is  used  some 
cleuteroalbumose  (Kiihne)  frequently  remains  unprecipitated ; 
the  disadvantage  consists  in  the  fact  that  the  removal  of  the 
ammonium  sulphate  afterwards  offers  far  greater  difficulties 
than  that  of  the  sodium  chloride. 

Separation  of  the  Albumoses  from  Peptone  by  Means  of 
Ammonium  Sulphate. 

Put  the  solution  evaporated  to  200  cc.  into  a  large  mortar 
containing  100  g.  of  finely  ground  ammonium  sulphate. 
Grind  thoroughly  until  the  ammonium  sulphate  is  all  dis- 
solved and  separate  the  viscous  mass  of  albumoses,  which 
precipitates,  from  the  solution  by  decantation.  The  solution 
is  kept ;  the  precipitate  is  ground  once  more  with  a  solution 

^  By  precipitating  a  solution  of  this  kind  with  alcohol  the  commercial 
peptone  (mixture  of  albumoses  and  peptone)  is  prepared. 


GASTRIC  DIGESTION.  43 

of  ammonium  sulphate,  separated  from  the  solution,  and  this 
second  solution  then  thrown  away. 

It  is  now  necessary  to  free  the  albumose  precipitate  from 
the  ammonium  sulphate  clinging  to  it  and  inclosed  in  it.  For 
this  purpose  dissolve  it  in  water  and  boil  the  tolerably  dilute 
solution  (best  in  an  enamelled-iron  dish)  with  barium  car- 
bonate, replacing  the  water  which  evaporates  by  hot  water. 
By  boihng  with  barium  carbonate  the  sulphuric  acid  is  com- 
bined vnth  the  barium  and  the  ammonia  escapes.  Continue 
the  boiling  until  the  fluid  no  longer  smells  of  ammonia  and 
a  filtered  portion  gives  no  cloudiness  with  barium  chloride 
solution  and  therefore  all  the  sulphuric  acid  is  united  to  the 
barium.  Then  filter.^  The  albumose  solution  thus  obtained 
very  frequently,  perhaps  always,  contains  barium,  often  in 
considerable  amount  (shown  by  adding  dilute  sulphuric  acid 
to  a  small  portion  of  the  solution).  To  separate  the  barimn 
add  ammonia  and  ammonium  carbonate  as  long  as  a  precipi- 
tate is  formed,  heat,  and  filter  from  the  barium  carbonate, 
best  after  allowing  to  stand  for  some  time.  Evaporate  the 
filtrate  on  the  water-bath  to  a  small  volume  and  precipitate 
with  95  per  cent,  or  absolute  alcohol.  The  albumoses  pre- 
cipitate in  the  form  of  a  viscous  mass.  Let  stand  for  some 
hours  under  the  strong  alcohol,  best  with  renewal  of  the 
alcohol,  until  the  mass  has  become  hard  and  brittle,  pour  off 
the  alcohol,  grind  the  residue  in  a  mortar  with  absolute  alco- 
hol, bring  the  whole  mass  into  a  vessel  which  may  be  closed, 
let  stand  in  this  for  twenty-four  hours,  filter,  and  wash  with 
ether.  We  thus  obtain  a  fine  white  or  yellowish- white  pow- 
der, which  according  to  Kiihne  is  a  mixture  of  four  substances: 
dysalbumose,   protalbumose,   heteroalbumose,   and   deutero- 

'  The  filtrate  should  be  clear ;  however,  a  slight  cloudiness  does  not 
matter  in  case  the  solution  is  afterwards  to  be  treated  with  ammonium 
carbonate,  as  the  precipitate  of  barium  carbonate  thereby  formed  carries 
down  with  it  the  remainder  of  the  barium  sulphate. 


44     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

albumose.^  The  separation  of  these  bodies  will  not  be  taken 
up  here.  The  first  three  of  these  albumoses  are  also  called 
primary  albumoses  in  contradistinction  to  deuteroalbumose. 

(a)  Conduct  of  Albumose  on  Heating.  Heat  a  small  por- 
tion for  three  hours  to  130°  to  140°,  let  cool,  and  treat  with 
water.  The  albumose  now  only  partly  dissolves  in  water. 
Filter  and  wash.  Warm  the  insoluble  residue  in  a  test-tube 
with  dilute  soda  solution:  partial  solution.  Acidify  the 
filtered  solution  cautiously  with  hydrochloric  acid:  precipi- 
tate. The  albumose  is  reconverted  by  heating  into  a  body 
resembling  albumin,  presumably  by  the  loss  of  water  (R. 
Hofmeister). 

(6)  Dissolve  5  g.  of  the  albumose  by  warming  with  100  cc. 
of  water.  The  solution  formed  is  turbid  (dysalbumose  and 
residue  of  albumin).  The  filtered  solution  is  used  for  the 
following  reactions: 

Reactions  of  Albumose. 

1,  Heat  a  small  portion  of  the  solution  to  boiling:  it 
remains  unchanged  (after  transient  cloudiness)  even  after 
acidifying  with  acetic  acid  and  adding  a  very  few  drops  of 
sodium  chloride  solution. 

2,  Acidify  with  acetic  acid  and  add  some  concentrated 
sodium  chloride  solution:  the  solution  becomes  cloudy,  but 
clears  up  on  heating.     On  cooling  it  again  becomes  cloudy. 

3,  Add  to  a  small  portion  of  the  solution  a  few  drops  of 
nitric  acid;  a  cloudiness  or  precipitate  ^  soluble  in  an  excess 
of  the  nitric  acid  results.     The  solution  becomes  lemon-yellow 

^  According  to  the  investigations  of  R.  Hofmeister  and  his  pupils  these 
substances  are  also  in  part  not  simple  but  mixtures. 

^  If  the  solution  contains  but  little  sodium  chloride,  the  cloudiness 
may  not  appear.  Repeat  the  experiment,  in  this  case  adding  some  salt 
solution  before  treating  "with  the  nitric  acid.  If  the  albumose  is  obtained 
from  the  meat  residue,  this  reaction  only  takes  place  after  the  addition 
of  sodium  chloride. 


GASTRIC  DIGESTION.  45 

on  standing  or  gently  warming;  on  supersaturating  with 
caustic  soda  this  color  changes  to  orange  (xanthoproteic 
reaction) . 

4.  Acidify  the  solution  with  a  few  drops  of  acetic  acid  and 
then  add  some  potassium  ferrocyanide  solution:  marked 
cloudiness  which  disappears  on  heating  (often  not  com- 
pletely). 

5.  Add  to  a  portion  of  the  solution  half  its  volmne  of 
caustic  soda  and  then,  drop  by  drop,  a  dilute  solution 
of  copper  sulphate.  The  copper  hydroxide,  which  first 
precipitates,  dissolves  on  shaking  with  a  purplish-violet  color : 
biuret  reaction.  An  excess  of  copper  sulphate  changes  the 
color  of  the  solution  to  a  blue  violet.  The  biuret  reaction  is 
not  characteristic  of  albumoses,  as  albumin  also  gives  it. 

A  part  of  the  albumose  solution  is  then  diluted  to  ten 
times  its  volume  (0.5  per  cent.).  "" 

1.  Biuret  Reaction,  Posner's  Modification.  Float  a  dilute 
solution  of  copper  sulphate  on  the  diluted  albumose  solution, 
containing  half  its  volume  of  sodium  hydroxide  solution  (so 
that  the  copper  sulphate  solution  runs  down  the  wall  of  the 
tube,  held  in  an  inchned  position,  and  the  fluids  do  not  mix). 
The  characteristic  color  develops  at  the  surface  of  contact  of 
the  two  fluids  or  proceeds  from  this.  Recommended  for 
dilute  solutions.  With  such  solutions  w^e  may  also  use  to 
advantage  an  ammoniacal  solution  of  copper  sulphate  or 
Fehhng's  solution. 

2.  Add  to  small  portions  of  the  solution  (1)  mercuric 
chloride  solution,  (2)  a  solution  of  tannin,  (3)  some  drops  of 
hydrochloric  and  phosphotungstic  acids:  insoluble  precipi- 
tates. 

3.  Add  to  a  small  portion  of  the  solution  a  few  drops  of 
Millon's  reagent  and  heat:  red  precipitate. 


46     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

Preparation  of  Peptone. 

The  solution  obtained  in  the  precipitation  of  the  albu- 
moses  contains  peptone  together  with  traces  of  albumoses, 
especially  deuteroalbumose.  Dissolve,  by  heating,  20  g.  more 
of  ammonium  sulphate  in  this  solution,  or  so  much  of  it  as 
may  be  necessary  to  form  a  saturated  solution,  and  make 
alkaline  with  ammonia  and  ammonium  carbonate.  After 
cooling,  filter,  heat  until  the  odor  of  ammonia  has  disappeared, 
saturate  again  at  the  boiling-point  with  ammonium  sulphate, 
let  cool,  and  when  perfectly  cold  filter  from  the  ammonium 
sulphate  and  traces  of  albumose  which  separate.^  Before 
preparing  peptone  from  the  filtrate  determine  by  the  biuret 
reaction  whether  it  contains  any  considerable  quantity  of 
peptone.  For  this  purpose  add  to  a  portion  of  the  filtrate 
so  much  caustic  soda  solution  of  1.34  specific  gravity  that 
sodium  sulphate  begins  to  separate,  then  add  the  copper 
sulphate  solution.  If  an  intensely  red  fluid  does  not  result, 
further  work  with  the  solution  will  not  pay.  The  removal 
of  ammonium  sulphate  from  the  solution  is  accomplished  in 
the  same  manner  as  with  albumose,  by  boihng  with  bariimi 
carbonate,  etc.  It  is  advisable,  however,  before  treating 
with  barium  carbonate  to  separate  a  part  of  the  ammonium 
sulphate  by  evaporation  and  crystalhzation  and  also  by  pre- 
cipitation with  alcohol,  in  which  the  peptone  dissolves.  Since 
the  barium  carbonate  as  a  rule  is  not  entirely  free  from  solu- 
ble salts,  these  together  with  the  sodium  chloride  accumulate 
in  the  fluid  containing  the  peptone,  so  that  the  peptone 
obtained  always  yields  considerable  ash. 

The  reactions  are  the  same  as  those  of  albumose,  but  no 

^  To  effect  a  complete  separation,  the  solution  made  acid  with  acetic 
acid  must  be  again  saturated  with  ammonium  sulphate  at  the  boiling- 
point,  allowed  to  cool,  and  filtered  once  more.  Kiihne,  Zeitschr.  f.  Biol. 
29,  1  (1892).— 0. 


GASTRIC  DIGESTION.  47 

precipitates  are  given  by  acetic  acid  and  potassium  ferro- 
cyanide,  acetic  acid  and  sodium  chloride  or  by  nitric  acid. 

Instead  of  using  ammonium  sulphate  we  may  also  precipitate  the 
albumoses  from  the  acidified  solution  by  means  of  sodium  chloride. 
For  this  purpose  add  to  the  200  cc.  of  the  solution  obtained  10  cc.  of 
glacial  acetic  acid  and  grind  the  mixture  with  75  g.  of  pure  sodium 
chloride.  The  separation  takes  place  the  same  as  it  did  when  am- 
monium sulphate  was  used :  wash  the  albumoses  with  a  concentrated 
solution  of  sodium  chloride.  The  sodium  chloride  is  removed  by 
dialysis.  After  the  dialysis  concentrate  the  solution  and  precipitate 
mth  alcohol.  We  may  also  purify  the  precij^itate  by  dissolving  it 
in  water,  heating  the  solution,  adding  a  solution  of  salt  until  the  hot 
fluid  is  no  longer  clear,  cooling  and  purifying  the  precipitate  thus 
obtained  by  dialysis.  The  precipitation  of  the  albumose  by  acetic 
acid  and  salt  is  not  so  complete  as  that  vnth  ammonium  sulphate. 
The  peptone  remaining  in  solution  therefore  contains  albumose. 

According  to  S.  FrankeP  albumose  and  peptone  may  be  separated 
from  each  other  by  means  of  alcohol  alone. 

1  Wiener,  med.  Blatter,  1896,  Nos.  45  and  46. 


CHAPTER  IV. 
EXAMINATION  OF  BLOOD. 

(a)  Defibrinated  Blood. 
I.  Alkaline  Reaction  of  Blood. 
II.  Reaction  with  Guaiacum  and  OU.  of  Turpentine. 

III.  Conduct  towards  Hydrogen  Peroxide. 

IV.  Solution  of  the  Blood-corpuscles. 
V.  Crystallized  Oxyhsemoglobin. 

VI,  Spectroscopic  Examination  of  Oxyhsemoglobin,  Hsemoglobin, 

Methsemoglobin,  and  Sulphohsemoglobin. 
VII.  Carbon  Monoxide  Hgemoglobin. 
^QIL  Alkaline  Haematin  Solution.     Reduced  Hsematin. 
IX.  Hsematin  Hydrochloride. 
X.  Hsemin  Test. 
XI.  Hsematoporphyrin. 
XII.  Conduct  of  Blood  on  Heating. 

(b)  Blood-fibrin. 

I.  Conduct  towards  the  0.27  per  cent.  Hydrochloric  Acid. 
II.  Conduct  towards  Hydrogen  Peroxide. 
III.  Conduct  towards  Neutral  Salts. 

(c)  Blood-se*rum. 
I.  Precipitation  of  the  Albmnin  by  Salts. 

II.  Separation  of  the  Proteids. 
III.  Reactions  of  the  Proteids  of  the  Blood-serum. 

(a)  Defibrinated  Blood. 
I.  Reaction  of  Blood. 

The  alkaline  reaction  of  blood  cannot  be  shown  with  ordi- 
nary litmus  paper  alone,  since  this  becomes  saturated  with 

48 


EXAMINATION  OF  BLOOD.  49 

the  blood-pigment  or  blood-corpuscles.  But  the  reaction 
may  be  shown  very  beautifully  if  we  allow  a  few  drops  of 
violet-red  litmus  tincture  to  soak  into  a  porous  clay  plate, 
and  then  place  upon  this  spot  a  drop  of  blood  and  wash  it 
off  at  once.  There  is  thus  obtained  a  distinct,  even  intensely 
blue  spot  (Liebreich).  But  even  with  ordinary  litmus  paper 
the  alkahne  reaction  may  be  shown  in  the  following  manner: 
Grind  some  blood  in  a  mortar  with  an  excess  of  powdered 
ammonium  sulphate,  so  that  even  after  long  grinding  a  por- 
tion of  the  salt  still  remains  undissolved.  Into  this  paste 
dip  a  rather  wide  strip  of  red  litmus  paper  and  then  wash  it 
off  thoroughly  with  water. — Zuntz  recommends  litmus  paper 
made  out  of  tissue-paper.  Moisten  this  with  a  concentrated 
solution  of  salt  or  sodium  sulphate  or  magnesium  sulphate, 
place  a  small  drop  of  blood  on  the  paper  with  a  glass  rod,  and 
absorb  the  liquid  at  once  with  blotting-paper. 

II.  Conduct  towards  Guaiacum  and  Oil  of  Turpentine. 

To  8  or  10  cc.  of  water  add  a  few  drops  of  blood  (shaking 
thoroughly),  then  some  tincture  of  guaiacum  (freshly  pre- 
pared by  dissolving  some  gum  guaiacum  in  alcohol  in  a  test- 
tubo)  till  the  solution  becomes  milky,  and  finally  some  oil  of 
turpentine  which  has  stood  for  some  time.  On  shaking,  the 
mixture  becomes  intensely  blue  (oxidation  of  the  gum  guaia- 
cuni:  the  blood-corpuscles  act  as  carriers  of  oxygen  from  the 
ozonized  oil  of  turpentine  to  the  gum  guaiacum). 

III.  Reaction  with  Hydrogen  Peroxide. 

To  a  few  cubic  centimeters  of  blood  add  several  times  the 
volume  of  a  solution  of  hydrogen  peroxide :  marked  frothing 
due  to  the  escape  of  oxygen  (so-called  catalytic  action  of  the 
blood-pigment). 


50     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

IV.  Solution  of  the  Blood-corpuscles. 

Treat  a  small  portion  of  the  blood  in  a  test-tube  with  some 
ether  and  some  water,  and  shake  thoroughly:  the  blood-cor- 
puscles dissolve,  the  blood  becomes  transparent  (laky  blood). 
A  solution  of  the  salts  of  the  bile-acids  acts  in  the  same  way 
on  the  blood. 

V.  Preparation  of  Crystallized  Oxyhaemoglobin. 

The  preparation  of  crystallized  oxyhaemoglobin  is  readily 
accompUshed  only  with  certain  kinds  of  blood  (dog,  horse, 
guinea-pig,  rat),  but  not  with  the  blood  of  man,  ox,  pig,  or 
rabbit. 

One  hundred  cubic  centimeters  of  dog's  blood  are  shaken 
vigorously  in  a  flask  with  air,  cooled  to  0°,  shaken  with  10  cc. 
of  ether  and  10  cc.  of  water,  so  that  the  blood-corpuscles  dis- 


FiG.  3. — Crystals  of  Oxyhemoglobin  from  Dog's  Blood. 

solve  and  the  blood  becomes  laky  (the  complete  solution  of 
the  blood-corpuscles  is  to  be  determined  by  means  of  a  micro- 
scopical examination),  and  the  mixture  allowed  to  stand 
at  0°.  The  separation  of  the  oxyhsemoglobin  crystals  takes 
place  at  once  or  after  a  few  hours  when  dog's  blood  is  used. 


EXAMINATION  OF  BLOOD.  51 

Examine  under  the  microscope.  To  purify  the  oxy haemo- 
globin, filter  at  a  low  temperature,  press  out  the  mother- 
liquor,  dissolve  the  crystals  in  as  small  a  quantity  of  water 
■as  possible  at  30°,  filter  quickly,  add  one-fifth  to  one-fourth 
the  volume  of  alcohol  gradually  and  with  constant  stirring 
to  prevent  coagulation,  and  let  stand  at  0°  until  crystalHza- 
tion  is  complete. 

VI.  Spectroscopic  Examination.* 

1.  Oxyhaemoglobin  and  Haemoglobin.  Ten  cubic  centi- 
meters of  blood  are  diluted  to  100  cc,  filtered,  then  gradually 
further  diluted  until  the  solution  when  examined  spectro- 
scopically  shows  distinctly  the  oxyhsemoglobin  bands  between 
D  and  E  in  the  yellow  and  green  of  the  spectrum  (see  Table 
of  Absorption  Spectra, ^  No.  1).  Then  add  to  the  solution  a 
few  drops  of  the  so-called  Stokes's  solution  (solution  of  fer- 
rous ammonium  tartrate,  which  must  always  be  freshly 
prepared,  made  by  dissolving  in  water  a  piece  of  ferrous  sul- 
phate as  big  as  a  pea,  adding  as  much  tartaric  acid  as  may 
be  held  on  the  point  of  a  knife-blade  and  then  ammonia 
to  alkahne  reaction:  clear  greenish  solution).  The  blood 
changes  its  color  at  once,  becomes  bluish  or  violet,  and  shows 
in  place  of  the  two  absorption-bands  a  broad  band  due  to 
haemoglobin  (No.  2  in  the  Table  of  Absorption  Spectra).  The 
reduction  may  also  be  brought  about  by  adding  a  few  drops 
of  ammonium  sulphide  solution  and  allowing  the  mixture 
to  stand  for  some  minutes;  but  this  takes  longer  and  in 
addition  to  the  broad  band  of  the  haemoglobin  there  always 
appears  a  weaker  and  narrower  band  in  the  red,  which  pre- 
sumably comes  from  the  sulphoha^moglobin  compound. 

•  For  most  work  in  physiological  chemistry  the  Browning  pocket  spec- 
troscope is  to  be  preferred  on  account  of  its  convenience. — O. 

^  For  a  more  recent  taljle  of  absorption  spectra,  see  Ziemke  and  Miiller, 
Archiv  fiir  Anat.  u.  Physiol.,  1901,  Sp.  Bd.  177.— O. 


52     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

2.  Methaemoglobin.  To  prepare  a  solution  of  methsemo- 
globin,  add  to  some  of  the  same  or  a  more  concentrated  solu- 
tion of  blood  a  few  drops  of  a  strong  solution  of  potassium 
ferricyanide  (freshly  prepared).  The  solution  turns  brown 
and  shows  a  characteristic  spectrum  (No.  3  in  the  Table  of 
Absorption  Spectra).  Especially  to  be  noted  is  the  strong 
absorption  of  light  in  the  blue  part  of  the  spectrum.  Now 
add  to  the  methsemoglobin  solution  a  few  drops  of  ammonium 
sulphide,  let  stand  some  minutes,  and  then  shake  vigorously 
with  air.  The  solution  now  shows  the  bands  of  oxyhsemo- 
globin.  The  methaemoglobin  may  therefore  be  converted  by 
reduction  and  subsequent  oxidation  into  oxy haemoglobin. 

3.  Sulphohaemoglobin.  Conduct  hydrogen  sulphide  into 
the  blood  solution,  which  has  been  previously  thoroughly 
shaken  with  air.  It  turns  brown,  then  dirty  green,  due  to 
the  formation  of  sulphohaemoglobin,  and  when  examined  with 
the  spectroscope  shows  an  absorption-band  between  the  yel- 
low and  orange  near  the  C  line  (E.  Harnack). 

VII.  Carbon  Monoxide  Haemoglobin. 

Conduct  illuminating-gas  (or  carbon  monoxide)  into  50  cc. 
of  blood  until  it  becomes  cUstinctly  cherry-red.  When  exam- 
ined with  the  spectroscope  at  the  proper  dilution  it  gives 
almost  exactly  the  same  absorption-bands  as  the  oxyhaemo- 
globin,  only  they  are  a  little  nearer  the  violet  end  of  the  spec- 
trum. On  the  addition  of  ammonium  sulphide  or  Stokes's 
solution  no  reduction  takes  place,  however,  and  the  bands 
remain  unchanged. 

To  distinguish  carbon  monoxide  haemoglobin  from  oxy- 
haemoglobin  or  to  detect  the  former  in  the  presence  of  the 
latter,  a  great  number  of  reactions  have  been  given,  all  of 
which  depend  upon  the  greater  stabiUty  of  the  carbon  mon- 
oxide haemoglobin  (the  detection  of  carbon  monoxide  haemo- 
globin in  mixtures  spectroscopically  is  difficult  and  only  pos- 
sible to  a  certain  extent). 


EXAMINATION  OF  BLOOD.  53 

(a)  Test  of  Katayama.  Add  5  drops  of  blood  containing 
carbon  monoxide  to  10  cc.  of  water,  then  add  5  drops  of 
orange-colored  ammonium  sulphide  and,  after  mixing,  10 
drops  of  acetic  acid  or  as  much  as  may  be  necessary  to  make 
the  mixture  faintly  acid.  With  the  blood  containing  carbon 
monoxide  a  rose-red  color  appears,  with  the  normal  blood  a 
dirty  greenish-gray  color.  The  color  is  still  perceptible  with 
one  part  of  the  carbon  monoxide  blood  to  five  of  normal  blood. 

(b)  Test  of  Kunkel.  Mix  carbon  monoxide  blood  with 
four  times  its  volume  of  water.  To  a  measured  quantity  of 
this  mixture  add  an  equal  volume  of  a  3  per  cent,  tannin 
solution.  Repeat  with  normal  blood  and  note  any  difference, 
especially  on  standing. 

(c)  Rubner's  Test.  Add  to  the  undiluted  blood  four  to 
five  times  the  volume  of  basic  lead  acetate  solution  and  shake 
thoroughly  for  a  minute.  The  carbon  monoxide  blood  turns 
a  beautiful  red,  the  normal  blood  a  brown.  After  standing 
for  some  time  the  normal  blood  gradually  becomes  a  chocolate 
and  brownish  gray,  while  the  carbon  monoxide  blood  remains 
unchanged. 

The  tests  (6)  and  (c)  are  especially  distinct  when  the  mix- 
tures are  allowed  to  stand  for  some  time  in  the  test-tubes. 
They  show  carbon  monoxide  blood  in  the  presence  of  normal 
blood,  in  the  proportion  of  1  to  6  or  even  1  to  10.  They  are 
therefore  especially  suited  to  detect  small  quantities  of  carbon 
monoxide  in  blood. 

VIII.  Alkaline  Haematin  Solution. 

By  heating  as  well  as  by  the  action  of  alkalies  or  acids  the 
haemoglobin  is  split  up  into  coagulated  albumin  and  a  pigment. 
The  pigment  is  different,  according  as  hjcmoglobin  is  decom- 
posed in  the  absence  of  oxygen  or  oxyhemoglobin  in  the 
presence  of  oxygen  (air).  In  the  first  case  reduced  hsema- 
tin  (ha^mochromogen  of  Hoppe-Seyler)  is  formed,  in  the 
latter  haematin  or  this  together  with  ha^mochromogen.    This 


54    PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

cleavage  takes  place  not  only  in  solutions  of  haemoglobin,  but 
also  in  blood  itself. 

To  show  the  formation  of  hsematin  add  about  1  cc.  of 
caustic  soda  solution  to  8  or  10  cc.  of  diluted  blood  (1 :  5),  and 
heat :  the  solution,  which  at  first  is  almost  cherry-red,  turns 
brown-green.  When  examined  with  the  spectroscope  the 
Hght  is  found  to  be  entirely  absorbed  up  to  a  part  of  the  red. 
The  spectrum,  on  further  dilution,  is  not  very  characteristic; 
at  the  proper  concentration  it  consists  of  a  broad,  poorly 
defined  band  in  the  orange  between  C  and  D.  On  the  addi- 
tion of  one  to  two  drops  of  ammonium  sulphide  or  Stokes's 
solution  this  absorption-band  disappears  and  the  two  sharply 
defined  and  intense  absorption-bands  of  the  reduced  hsematin 
or  hsemochromogen  (No.  5  in  the  Table  of  Absorption  Spectra) 
appear.  These  have  approximately  the  same  position  as 
those  of  the  oxyhsemoglobin,  but  lie  nearer  to  the  violet  end  of 
the  spectrum.  The  band  nearer  to  the  red  is  narrower  and 
more  sharply  defined,  that  towards  the  violet  is  broader,  less 
intense,  and  not  so  sharply  defined. 

IX.  Hsemin  ^  (Haematin  Hydrochloride),  CgjHg^ClN^FeOg. 

Small  quantities  of  hsemin  are  most  readily  obtained  in  the 
following  manner:  75  cc.  of  glacial  acetic  acid,  previously 
saturated  with  salt,, are  heated  in  a  flask  on  the  water-bath 
to  90°,  then  25  cc.  of  blood  are  added  quite  gradually  and 
with  constant  shaking;  continue  the  heating  at  90°  for  about 
ten  minutes,  then  pour  into  a  beaker  and  let  stand  for  twenty- 
four  hours.  The  hsemin  will  be  found  on  the  bottom  of  the 
beaker  in  the  form  of  an  intensely  bluish-black  layer  of  ght- 
tering  crystals.     (Examine  under  the  microscope.) 

The  supernatant  fluid  is  siphoned  off,  the  crystals  are 
washed  once  with  glacial  acetic  acid,  then  with  acetic  acid 

^  Nencki  calls  this  substance  acethsemin  and  gives  it  the  formula 
Cs^HaaNiFeClO^.— O. 


EXAMINATION  OF  BLOOD.  55 

and  water,  and  filtered.  From  the  hsemin  the  hff'matin  may 
be  obtained  by  dissolving  it  in  dilute  caustic  soda  solution, 
precipitating  with  dilute  hydrochloric  acid,  filtering,  and 
washing.  Since  the  quantity  of  the  ha-matin  thus  obtained 
is  very  small  and  sticks  to  the  filter,  it  is  advisable  to  dissolve 
it  by  pouring  on  the  filter  a  solution  of  ammonia,  and  then  to 


Fig.  4. — Hsematin  Hydrochloride,  Hsemin  Cr\'stals. 
evaporate  this   solution   to  dryness  on  the  water-bath.     On 
incinerating,  the  hsematin  leaves  the  red  oxide  of  iron  (13.5 
per  cent.). 

X.  Haemin  Test. 

The  formation  of  hsematin  hydrochloride  is  a  very  excel- 
lent means  of  recognizing  blood-stains.  Some  blood,  dried 
in  the  air  but  not  heated,  is  ground  in  a  mortar  with  a  trace 
of  salt,  then  boiled  in  a  dry  test-tube  with  glacial  acetic  acid 
and  the  fluid  obtained  evaporated  in  a  watch-glass  on  a 
water-bath  heated  not  quite  to  boiling.  The  test  may  also 
be  made  on  a  microscope-slide.  Crush  some  of  the  dry  blood 
with  a  knife-blade,  mix  it  with  some  salt,  put  the  cover-glass 
on,  let  some  glacial  acetic  acid  flow  under  this,  and  heat  the 
slide  over  a  very  small  luminous  flame  till  the  liquid  just 
begias  to  boil,  then  let  some  more  glacial  acetic  acid  flow  on 


56     PHYSIOLOGICAL  AND  PATHOLOGICAL   CHEMISTRY. 

from  the  rim,  heat  again  and,  after  coohng,  examine  the  sUde 
under  the  microscope.  In  case  no  hsemin  crystals  are  found, 
examine  again  after  allowing  to  stand  for  a  longer  time.  We 
may  also  let  a  drop  of  blood  dry  on  a  piece  of  muslin,  then 
cut  out  the  spot  and  boil  this  with  glacial  acetic  acid,  etc. 

XI.  Hsematoporphyrin. 

Hsematoporphyrin,  according  to  Nencki  and  Sieber, 
CifiHigNsOg,^  is  formed  from  hsematin  by  warming  the  solution 
of  hsemin  in  glacial  acetic  acid  saturated  with  hyclrobromic 
acid  gas,  according  to  the  equation  ^ 

C32H32N4FeO,+ 2HBr  +  2H2O  =  2CieHi8N203  +  FeBr2+  H^. 

The  anhydride,  CgjHg^N^s,  is  formed  by  the  action  of  concen- 
trated sulphuric  acid  upon  htematin  or  haemoglobin.  Add  to 
8  or  10  cc.  of  concentrated  sulphuric  acid,  drop  by  drop,  and 
with  constant  shaking,  five  drops  of  blood.  The  clear  red- 
violet  solution  which  results  shows  when  examined  with  the 
spectroscope  two  very  beautiful  and  characteristic  absorp- 
tion-bands: a  narrow  band  in  the  orange  and  a  broader  one 
in  the  yellow  and  green  (No.  6  in  the  Table  of  Absorption 
Spectra).  These  show  some  resemblance  to  the  bands  of 
oxyhaemoglobin,  but  lie  nearer  to  the  red  end  of  the  spectrum. 
The  broad  band  is  especially  characteristic  in  that  it  consists 
of  two  parts,  a  less  intense  part  towards  the  narrow  band  and 
a  deep-black  part  on  the  other  side;  frequently  the  less  in- 
tense part  of  the  broad  band  shows  an  edge  towards  the  red, 
which  is  marked  by  stronger  absorption,  so  that  we  may  also 
speak  of  three  absorption-bands  in  the  spectrum. 

^  According  to  Zaleski  the  formula  for  hsematoporphyrin  hydrochloride 
is  Cj^HjgOjN^  •  2HC1,  and  the  formation  of  hsematoporphyrin  from  hsemin 
is  represented  by  the  following  equation: 

Cg.Ha.O^N.FeCl  +  2HBr  +  2H2O  =  Cs.HgsOaN^  +  FeBr^  +  HCl. 
Zeitschr.  f.  physiol.  Chem.  37,  74.— O. 


EXAMINATION  OF  BLOOD.  57 

XII.  Coagulation  of  the  Blood  by  Heating. 
Diluted  blood  heated  to  boiling  and  filtered. 


Colorless  filtrate,  tested  for  Coagulum,    colored    brown    from 

sugar  and  salts.  hsematin. 

In  every  investigation  of  the  soluble  constituents  of  the 
blood  it  is  necessary  first  to  precipitate  the  proteids.  This  is 
accomplished  either  by  pouring  the  blood  into  four  times  its 
volume  of  absolute  alcohol  or  by  heating  to  boiling. 

Heat  a  mixture  of  30  to  50  cc.  of  blood  and  six  to  eight 
times  its  volume  of  water  over  a  free  flame,  and  with  constant 
stirring,  to  \dgorous  boihng,  taking  care  to  keep  the  reaction 
neutral  or  very  faintly  acid  by  the  cautious  addition  of  dilute 
acetic  or  sulphuric  acids,  then  filter.  The  filtrate  should  be 
clear  and  colorless,  which  is  only  possible  with  fresh  blood. 
The  solution  is  evaporated  to  a  small  volume,  then  divided 
into  halves,  one  of  which  is  used  to  perform  Trommer's  test 
with  some  freshly  mixed  Fehhng's  solution,  the  other  is 
further  evaporated  on  the  water-bath,  a  drop  allowed  to 
evaporate  on  a  microscope-sHde  and  examined  under  the 
microscope:  crystals  of  sodium  chloride.  Evaporate  the 
residue  to  dryness  and  incinerate:  salts,  especially  sodium 
chloride.  Test  for  chlorides  with  silver  nitrate,  for  phos- 
phates with  ammonium  molybdate.  The  test  for  sugar  fre- 
quently does  not  succeed  with  commercial  blood. 

The  brown-colored  coagulum  obtained  is  washed,  well 
pressed,  ground  in  a  mortar  with  100  cc.  of  absolute  alcohol, 
3  to  5  cc.  of  concentrated  sulphuric  acid  gradually  added, 
ground  again,  then  the  mixture  is  placed  in  a  flask  and  heated 
on  the  water-bath.  A  brown-colored  solution  results  and  an 
almost  colorless  residue  of  coagulated  albumin.  Filter,  and 
examine  the  filtrate  spectroscopically  (No.  4  in  the  Table  of 
Absorption  Spectra).     Especially  characteristic  is  the  band 


58     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

in  the  red  close  to  the  line  C,  Heat  the  solution,  after  adding 
some  tin  and  hydrochloric  acid,  in  a  flask  on  the  water-bath. 
The  solution  turns  yellowish-red  and  when  examined  spec- 
troscopically  shows  a  dark  band,  usually  not  sharply  defined, 
between  the  green  and  the  blue,  similar  to  that  of  urobiUn. 
Sometimes,  however,  only  a  diffuse  darkening  in  that  part  of 
the  spectrum  is  apparent. 

The  same  pigment  appears  to  be  present  sometimes  in 
urine ;  at  least  many  urines  show  similar  absorption  phenom- 
ena.- 

(6)  Blood-fibrin. 

I.  Conduct  towards  0.27  per  cent.  Hydrochloric  Acid. 

Pour  on  a  few  shreds  of  fresh  fibrin  (or  fibrin  kept  in 
glycerin,  after  it  has  been  well  washed)  some  0.27  per  cent, 
hydrochloric  acid  (3  cc.  of  hydrochloric  acid,  sp.  gr.  1.19,  to 
500  cc.  of  water).  The  fibrin  swells  gradually  and  dissolves 
on  long  digestion  at  40°  with  the  formation  of  acid  albumin. 
This  precipitates  on  neutrahzing  with  sodium  carbonate  solu- 
tion. 

II.  Conduct  towards  Hydrogen  Peroxide. 

Pour  on  a  few  shreds  of  fresh  fibrin  some  hydrogen  perox- 
ide solution:  evolution  of  oxygen.  Repeat  the  experiment 
with  boiled  fibrin:  no  gas  is  evolved.  The  action  of  the 
fibrin  on  the  hydrogen  peroxide  is  probably  due  to  the  pres- 
ence of  leucocytes  in  the  fibrin. 

III.  Conduct  towards  Salts. 

Fresh  fibrin  swells  and  gradually  dissolves  more  or  less 
completely  in  a  solution  of  potassium  nitrate. 

(c)  Blood-serum. 

The  blood-serum,  as  well  as  the  serous  fluids,  contains  a 
proteid  soluble  in  water,  serum  albumin,  and  one  insoluble 


EXAMINATIOX   OF  BLOOD.  59 

in  water,  serum  globulin,  kept  in  solution  by  the  salts  and 
the  alkali  of  the  serum. 

I.  Precipitation  of  the  Proteids  by  Salts. 

Grind  in  a  mortar  20  cc.  of  blood-serum  with  an  excess  of 
anunonium  sulphate  (about  15  g.)  for  a  considerable  time,  or 
repeat  the  process,  so  that  the  fluid  is  completely  saturated 
with  ammonium  sulphate:  by  this  means  both  the  proteids 
are  precipitated.  Filter  through  a  dry  filter.  The  filtrate 
is  perfectly  free  from  albumin;  when  heated  to  boihng  and 
acidified  with  acetic  acid  it  remains  clear. 

II.  Separation  of  Serum  Albumin  and  Serum  Globulin. 

Fifty  to  one  hundred  cubic  centimeters  of  blood-serum  (or 
serous  transudate)  are  treated  with  an  equal  volume  of  a 
saturated  solution  of  ammonium  sulphate,  filtered  and  washed 
with  a  half-saturated  solution  of  the  salt.  In  the  filtrate  the 
serum  albumin  will  be  found  (shown  by  heating  to  boiling) ; 
the  precipitate  is  serum  globulin.  It  dissolves  when  brought 
into  water  o\^dng  to  the  ammonium  sulphate  adhering  to  it; 
this  solution  coagulates  on  heating.  For  the  preparation  of 
pure  sermn  albumin  or  serum  globulin  this  method  is  not  suit- 
able, since  the  adhering  salts  can  only  be  separated  by  dialysis, 
and  the  removal  of  ammonium  sulphate  by  this  means  is  only 
accomphshed  vnth  great  difficulty.  Therefore  it  is  necessary 
to  saturate  the  blood  with  pulverized  magnesium  sulphate 
and  wash  the  precipitate  with  a  saturated  solution  of  the 
same  salt. 

Both  methods  are  generally  regarded-  as  being  of  equal 
value.  They  are,  however,  not  quite  eciuivalent:  the  quan- 
tity of  precipitate  obtained  by  the  use  of  ammonium  sulphate 
is  larger  than  that  with  magnesium  sulphate. 

The  precipitate  obtained  by  means  of  magnesium  sul- 
phate is  also  soluble  in  water  on  account  of  the  magnesium 


60     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

sulphate  adhering  to  it.     If  we  subject  the  solution  to  dialysis, 
part  of  the  globuUn^  separates.     This  is  washed  with  water. 

Suspended  in  water  it  dissolves  on  the  addition  of  a  trace 
of  caustic  soda  solution  and  separates  again  when  the  solu- 
tion is  exactly  neutralized  with  dilute  hydrochloric  acid. 
If  the  quantity  of  caustic  soda  used  to  dissolve  the  globulin 
was  too  great,  then  the  globulin  is  not  reprecipitated  on  neu- 
trahzation,  as  it  is  kept  in  solution  by  the  sodium  chloride 
formed. 

III.  Reactions  Common  to  Serum  Albumin  and 
Serum  Globulin. 

For  all  these  reactions  use  blood-serum  which  has  been 
diluted  with  four  times  its  volume  of  water  (20  cc.  of  blood- 
serum  diluted  to  100  cc.^). 

1.  A  small  portion  of  the  solution  when  heated  to  boihng 
changes  but  little;  it  becomes  somewhat  opaque  and  white 
by  reflected  light,  but  remains  transparent  by  transmitted 
light.  Coagulation  does  not  take  place  until  the  fluid  is  neu- 
traUzed  by  cautiously  adding  dilute  acetic  acid;  a  slight 
excess  of  acetic  acid  redissolves  the  precipitate.  The  solu- 
tion becomes  quite  clear  on  warming.  On  the  addition  of  a 
few  drops  of  concentrated  salt  solution  a  flocculent  precipi- 
tate of  albumin  is  formed. 

2.  To  a  little  of  the  solution  in  a  test-tube  add  about  one- 
half  the  volume  of  a  concentrated  solution  of  salt  and  divide 
into  two  approximately  equal  parts.  One-half  is  heated 
to  boiling:  coagulation  takes  place.  To  the  other  half  add 
acetic  acid  to  distinctly  acid  reaction:  it  becomes  cloudy  even 

^  The  quantity  of  the  insoluble  globulin  which  separates  is  always  very 
small;  according  to  recent  investigations  (Freund,  Marcus:  Zeitschr.  f. 
physiol.  Chem.  28,  559),  this  is  due  to  the  fact  that  globulin  is  for  the 
most  part  soluble  in  water. 

2  Correspondingly  diluted  pathological  transudates  (ascitic  or  dropsi- 
cal fluids)  may  also  be  used. 


EXAMINATION  OF  BLOOD.  61 

in  the  cold,  and  on  heating  gives  a  flocculent  precipitate. 
The  greater  the  amount  of  salt  in  the  albumin  solution  the 
less  is  the  coagulation  on  heating  dependent  upon  the  reac- 
tion of  the  fluid;  the  smaller  the  amount  of  salt  the  nearer 
must  the  reaction  be  to  neutral  or  faintly  acid  in  order  that 
coagulation  should  take  place  on  heating. 

3.  Add  nitric  acid  to  a  small  portion  of  the  solution:  a 
precipitate  forms  at  first,  which  disappears  on  shaking,  then 
on  the  addition  of  more  nitric  acid  a  permanent  precipitate  is 
formed,  which  does  not  dissolve  on  heating,  but  turns  yellow 
owing  to  the  formation  of  the  so-called  xanthoprotein. 

4.  If  we  add  glacial  acetic  acid  to  a  small  portion  of  the 
solution  and  heat,  acid  albumin  (acid  albuminate)  results. 
Cool  and  add  caustic  soda  solution.  While  the  reaction  is 
still  acid  a  precipitate  of  acid  albumin  forms,  soluble  in  an 
excess  of  caustic  soda. 

5.  Heat  a  Uttle  of  the  solution  with  half  its  volume  of 
caustic  soda  solution.  Alkah  albuminate  is  formed.  Cool 
and  neutralize  with  dilute  sulphuric  or  acetic  acid.  Albu- 
minate is  precipitated,  partially  soluble  on  heating  with  an 
excess  of  the  reagent. 

6.  If  we  add  copper  sulphate  solution  to  some  of  the  dilute 
blood-serum,  a  bluish-white  precipitate  of  copper  albuminate 
results,  which  dissolves  on  the  addition  of  caustic  soda  solu- 
tion to  a  deep-blue  fluid.  (The  salts  of  the  other  heavy 
metals  also  as  a  rule  give  precipitates.) 

7.  Add  some  mercuric  chloride  solution:  heavy  white 
precipitate  insoluble  in  an  excess  of  the  precipitating  reagent, 
but  soluble  in  concentrated  sodium  chloride  solution. 

8.  If  we  add  to  some  of  the  solution  a  few  drops  of  nitric 
acid  till  a  permanent  precipitate  is  formed  and  then  add 
absolute  alcohol  until  the  volume  of  the  mixture  has  been 
doubled,  the  greater  part  of  the  precipitate  dissolves  (dis- 
tinction from  egg-albumin). 


62     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

9.  When  concentrated  nitric  acid  of  1.48  specific  gravity 
is  added  to  some  of  the  solution,  the  precipitate  first  formed 
redissolves  to  a  clear  bright-yellow  fluid  as  soon  as  the  volume 
of  nitric  acid  added  is  equal  to  half  the  volume  of  the  albumin 
solution  (distinction  from  egg-albumin). 

10.  On  shaking  some  of  the  solution  with  an  equal  volume 
of  ether,  no  coagulation,  or  at  least  only  a  very  slight  one, 
results  (distinction  from  egg-albumin). 

11.  Add  to  some  of  the  solution  half  its  volume  of  caustic 
soda  of  1.34  specific  gravity  and  a  few  (3)  drops  of  a  neutral 
solution  of  lead  acetate  and  heat:  it  turns  brown  or  black 
(distinction  from  egg-albumin,  which  gives  a  much  darker 
solution) ;  when  acidified  with  hydrochloric  acid  there  is  soon 
obtained  a  grayish-yellow  cloudy  fluid  (distinction  from  egg- 
albumin).  The  reaction  depends  on  the  spHtting  off  of  sul- 
phur and  the  formation  of  lead  sulphide. 

Dilute  the  albumin  solution  with  nine  times  its  volume 
of  water  (10  cc.  diluted  to  100  cc.)  for  the  following  experi- 
ments: 

Reactions  of  very  Dilute  Solutions  of  Albumin. 

1.  Heat  to  boiling:  no  change.  Then  add  nitric  acid 
and  heat  again:   precipitation  of  coagulated  albumin. 

2.  Add  acetic  acid  and  potassium  ferrocyanide  solution: 
cloudiness  and  then  a  flocculent  precipitate. 

3.  Acidify  with  hydrochloric  acid  and  then  add  phospho- 
tungstic  acid:  voluminous  gelatinous  precipitate. 

4.  With  tannin  solution,  precipitation  takes  place,  and 
also, 

5.  With  mercuric  chloride  solution  (soluble  in  sodium 
chloride  solution). 

6.  Add  some  Millon's  reagent  and  heat  to  boiling:  coagu- 
lum,  which  gradually  turns  reddish  to  brick-red.    This  reac' 


EXAMINATION  OF  BLOOD.  63 

tion  depends  upon  the  presence  of  the  tyrosme  group  in  albu- 
min. It  is  also  given  by  all  benzene  derivatives,  which  con- 
tain a  hydroxyl  group  in  place  of  a  hydrogen  atom  of  the  ben- 
zene nucleus. 

For  the  reactions  of  coagulated  albumin  see  chapter  on 
Milk,  page  4. 


CHAPTER  V. 

PATHOLOGICAL  TRANSUDATES,  CYSTIC  FLUIDS. 

I.  Examination  for  Coagulable  Albumins. 

II.  Examination  for  Proteids  Precipitated  by  Acetic  Acid  and  In- 
soluble in  Excess  of  the  Reagent. 

III.  Examination  for  Albumin  and  Globulin. 

IV.  Examination  for  Urea.  , 
V.  Examination  for  Sugar. 

VI.  Examination  for  Paralbumin. 

I.  Examination  for  Coagulable  Albumins. 

See  in  this  connection  the  chapter  on  Blood,  page  48.  It 
is  to  be  noted  that  in  case  of  strongly  alkaline  transudates 
containing  but  little  albumin  no  coagulation  may  result  on 
heating.  In  such  cases  the  addition  of  the  acetic  acid  after 
the  heating  must  be  done  exceedingly  carefully,  and  in  case 
no  precipitate  appears  it  is  advisable  to  add  1  to  2  cc.  of  con- 
centrated salt  solution. 

II.  Proteids  Precipitated  by  Acetic  Acid.^ 

To  about  100  cc.  of  the  clear  fluid  add  acetic  acid  until  the 

reaction  is  distinctly  acid;  if  a  precipitate  insoluble  in  excess 

of  the  reagent  is  formed,  the  fluid  contains  mucin  or  nucleo- 

albumin.     Filter  off  the  precipitate,  wash,  then  grind  the 

^  In  case  pathological  fluids  are  not  available,  prepare  an  extract  from 
the  thymus  gland  of  the  calf  (1  part  of  the  finely  divided  gland  treated 
with  ten  parts  of  cold  water  for  24  hours  and  shaken  from  time  to  time 
and  then  filtered),  and  mix  this  with  an  equal  volume  of  blood-serum  or 
ascitic  (dropsical)  fluid. 

64 


PATHOLOGICAL  TRANSUDATES,  CYSTIC  FLUIDS.       65 

moist  precipitate  in  a  mortar  with  water,  adding  some  sodium 
carbonate  solution;  if  it  does  not  dissolve,  add  a  small  quan- 
tity of  caustic  soda  solution.  Filter,  precipitate  again  with 
acetic  acid,  and  wash  the  precipitate  with  water. 

To  distinguish  mucin  from  nucleoalbumin  we  may  use  the 
conduct  of  the  precipitate  on  heating  with  hydrochloric  acid 
(see  1  below) ;  mucin  forms  under  these  conditions  a  sub- 
stance which  reduces  copper  oxide  in  alkaline  solution,  while 
nucleoalbumin  does  not ;  nucleoalbumin  contains  phosphorus, 
while  mucin  does  not  (see  2  below).  To  test  for  phosphorus 
fuse  the  substance  with  soda  and  saltpeter,  whereby  the 
phosphorus  forms  alkali  phosphate.  The  presence  of  phos- 
phoric acid  in  the  fused  mass  is  only  to  be  regarded  as  proving 
the  presence  of  nucleoalbumin,  when  the  precipitated  proteid 
is  free  from  phosphates,  especially  calcium  and  magnesium 
phosphates,  and  from  the  very  widely  distributed  lecithin, 
which  contains  phosphorus. 

The  object  of  dissolving  the  precipitate  in  alkali  solution, 
filtering,  and  reprecipitating  with  acid  is  to  remove  the  phos- 
phates as  completely  as  possible. 

1.  Shake  half  of  the  moist  precipitate  of  proteid  with  a 
mixture  (about  25  cc.  or  somewhat  more)  of  three  volumes  of 
water  and  one  volume  of  hydrochloric  acid,  and  heat  in  a 
flask  on  the  wire  gauze  to  boiling,  or  heat  part  of  the  mixture 
in  a  test-tube.  Boil  gently  for  about  ten  minutes,  let  cool, 
make  a  portion  alkaline  with  caustic  soda  solution  without 
filtering,  then  test  with  Fehling's  solution,  or  add  a  small 
quantity  of  copper  sulphate  solution,  shake  thoroughly,  heat 
to  boiling,  and  cool  the  tube  by  placing  it  in  water:  in  the 
presence  of  mucin  red  cuprous  oxide  will  be  precipitated. 

2.  Grind  the  other  half  of  the  precipitate  in  a  mortar  with 
absolute  alcohol,  put  the  mixture  into  a  flask,  heat  on  the 
water-bath  to  boiling,  filter,  and  wash  with  some  alcohol. 
Press  the  moist  precipitate  between  filter-paper,  put  it  into 


66     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

a  dry  flask  and  pour  ether  over  it  (or,  better,  grind  it  in  a 
mortar  with  ether  and  then  put  the  mixture  into  a  flask), 
shake  vigorously,  let  stand  for  some  time,  then  filter,  and 
wash  with  ether.  Then  grind  0.3  to  0.5  g.  of  the  dry  precipi- 
tate with  thirty  times  its  weight  of  a  mixture  of  three  parts 
of  potassium  nitrate  and  one  part  of  sodium  carbonate  and 
fuse  the  mixture.  (See  in  this  connection  the  test  for  phos- 
phorus in  casein  in  the  chapter  on  Milk,  page  9.) 

The  presence  of  phosphoric  acid  in  the  fused  mass  shows 
that  we  have  to  do  with  nucleoalbumin.  It  is  advisable  to 
make  a  part  of  the  nitric  acid  solution  of  the  fused  mass  alka- 
line with  ammonia:  no  cloudiness  due  to  calcium  phosphate 
and  no  crystalline  precipitate  of  ammonium  magnesium  phos- 
phate should  appear.  Since,  however,  the  complete  removal 
of  calcium  phosphate  is  only  accomplished  with  great  diffi- 
culty, no  attention  should  be  paid  to  the  presence  of  a  trace 
of  phosphoric  acid  in  the  fused  mass.  This  may  come  from 
the  calcium  phosphate,  or  also  from  traces  of  adhering  lecithin. 
If  we  wish  to  be  quite  sure  of  the  absence  of  lecithin,  it  is 
recommended  to  treat  the  product  once  more  with  hot  abso- 
lute alcohol  before  fusing  it  with  the  oxidizing  mixture,  evap- 
orate the  alcoholic  extract  to  dryness,  and  fuse  the  residue 
with  soda  and  saltpeter.  In  this  fused  mass  there  must  be 
no  phosphoric  acid.^ 

III.  Examination  for  Serum  Albumin  and  Globulin. 

This  is  done  according  to  the  method  given  under  Blood- 
serum,  page  58. 

IV.  Examination  for  Urea. 

Exactly  neutralize  100  cc.  of  the  fluid  with  acetic  acid, 
then  pour  into  400  cc.  of  95  per  cent,  or  absolute  alcohol, 

^  In  regard  to  the  detection  of  the  xanthine  bases  and  the  pentose 
group,  which  is  present  in  very  many  nucleoalbumins,  see  the  chapter  on 
Pancreas,  page  76.  , 


PATHOLOGICAL  TRANSUDATES,  CYSTIC  FLUIDS.       67 

shake  or  stir  thoroughly,  and  after  twenty-four  hours  filter. 
Wash  the  coagulum  with  alcohol,  evaporate  the  filtrate  to 
drjTiess  at  a  low  temperature  on  the  water-bath,  dissolve  the 
residue  in  absolute  alcohol,  filter,  evaporate  to  dryness,  and 
again  dissolve  the  residue  in  ab'solute  alcohol.  If  it  now  dis- 
solves perfectly  clear,  evaporate  the  alcoholic  solution  to  dry- 
ness again ;  if  it  does  not,  the  treatment  with  absolute  alcohol 
is  repeated.  The  residue  obtained  by  the  evaporation  of  the 
alcohol  is  treated,  after  cooling,  with  a  few  drops  of  nitric  acid, 
and  allowed  to  stand  for  twenty-four  hours  in  the  cold. 
Usually  on  the  addition  of  the  nitric  acid  a  cloudiness  is  first 
formed,  caused  by  the  fatty  acids,  which  come  from  the  soaps 
almost  always  present;  gradually  urea  nitrate  crystaUizes 
out. 

If  we  vnsh  to  get  rid  of  these  fatty  acids,  which  contaminate  the 
urea  nitrate,  we  put  in  the  treatment  with  basic  lead  acetate.  The 
fluid  obtained  by  concentrating  the  first  alcoholic  extract,  which  is 
generally  turbid,  is  treated  with  a  solution  of  basic  lead  acetate,  drop 
by  drop,  as  long  as  the  precipitate  continues  perceptibly  to  increase, 
and  then  a  httle  ammonium  carbonate  solution  is  cautiously  added. 
The  fluid  above  the  flocculent  precipitate  now  becomes  quite  clear. 
Filter  and  pass  a  rapid  stream  of  hydrogen  sulphide  into  the  filtrate, 
filter  again  after  the  lead  sulphide  has  well  settled,  evaporate  the 
filtrate  to  dryness,  and  dissolve  the  residue  again  in  a  small  quantity 
of  absolute  alcohol,  etc. 

Examine  under  the  microscope  the  urea  nitrate  which 
separates  (compare  with  Fig.  7  in  the  chapter  on  Urine, 
page  95).  Then  after  completely  drying  the  crystals  by 
means  of  filter-paper  or  on  a  porous-clay  plate  and  washing 
them  with  some  ether,  dry,  and  heat  a  small  quantity  on 
platinum-foil  or  on  a  crucible-cover:  violent  decomposition 
or  explosion.  In  case  the  amount  of  the  urea  nitrate  suffices, 
convert  the  remainder  into  urea  (see  chapter  on  Urine,  page 
95),  and  test  this  by  means  of  its  reactions. 

The  amount  of  urea  contained  in  pathological  transudates 


68     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

and  exudates  is  very  small;  if  it  is  present  in  considerable 
quantities  it  indicates  a  direct  connection  of  the  fluids  with 
the  kidneys  or  urinary  ducts. 

If  the  test  for  urea  in  this  way  fails,  which  may  happen 
when  the  fluid  is  not  quite  fresh,  in  order  to  practice  the 
method  add  to  100  cc.  of  the  fluid  0.1  to  0.2  g.  of  urea  (pre- 
viously dissolved  in  water). 

Under  some  circumstances,  especially  when  the  organs  are  under 
examination  (in  retention  of  the  constituents  of  urine),  the  urea 
nitrate  may  be  mixed  with  hypoxanthine  nitrate :  this  admixture  may 
be  readily  detected  by  dissolving  a  weighed  quantity  of  the  urea 
nitrate  in  water  and  adding  ammonia  and  silver  nitrate.  Filter  off 
the  precipitate,  wash,  incinerate,  and  weigh.  We  thus  obtain  the 
weight  of  the  silver  corresponding  to  the  amount  of  hypoxanthine. 
From  this  the  amount  of  hypoxanthine  nitrate  may  be  calculated, 
and  this  is  then  subtracted  from  the  weight  of  urea  nitrate  taken. 

V.  Examination  for  Sugar. 

Proceed  at  first  just  as  in  the  examination  for  urea,  or 
coagulate  50  to  100  cc.  after  the  addition  of  a  few  drops  of 
acetic  acid  (in  case  the  fluid  contains  considerable  albumin  it 
is  to  be  diluted  with  one  or  more  times  its  volume  of  water), 
filter,  and  concentrate,  taking  care  that  the  reaction  does  not 
become  alkaline,  by  adding  a  few  drops  of  acetic  acid  when 
necessary.  With  the  solution  obtained,  amounting  to  about 
15  cc,  after  filtering  once  more,  try  the  a-naphthol  test,  the 
Trommer's  test,  and  also  the  fermentation  test  (see  in  this 
-connection  the  chapter  on  Urine,  "Detection  of  Sugar," 
page  118).  To  obtain  a  positive  result  with  the  fer- 
mentation test  usually  more  than  100  cc.  of  the  fluid  are 
necessary. 

VI.  Detection  of  Pseudomucin  (Paralbumin)  in  Cystic  Fluids. 

1.  Add  to  a  small  quantity  of  the  fluid  (about  25  cc.)  some 
drops  of  an  alcoholic  solution  of  rosolic  acid,  heat  to  boiling, 


PATHOLOGICAL  TRANSUDATES,  CYSTIC  FLUIDS.       69 

and  drop  in  very  dilute  sulphuric  acid  (tenth-normal  sul- 
phuric acid)  until  the  color  changes  to  yellow,  showing  that 
the  fluid  has  a  faintly  acid  reaction.  Heat  again  to  boiling 
and  filter:  in  the  presence  of  paralbumin  the  filtrate  will  be 
cloudy. 

2.  Precipitate  the  same  volume  (25  cc.)  of  the  cystic  fluid 
with  three  times  its  volume  of  95  per  cent,  alcohol,  filter, 
wash  a  few  times  with  alcohol,  press  the  precipitate  between 
filter-paper,  then  shake  it  thoroughly  with  a  mixture  of  one 
volume  of  hydrocliloric  acid  and  three  volumes  of  water,  and 
proceed  as  with  mucin  (see  page  71).  In  the  presence  of 
pseudomucin  we  obtain  a  precipitate  of  red  cuprous  oxide- 
Pseudomucin  is  not  precipitated  by  acetic  acid  and  is  thus 
distinguished  from  mucin. 

A  complication  may  arise  if  glycogen  is  also  present.  This 
ini<y  be  detected  by  treating  a  part  of  the  precipitate,  pro- 
duced by  alcohol,  with  water  and  saliva  and  testing  for  sugar. 
If  glycogen  is  present,  then  the  entire  quantity  of  the  pre- 
cipitate is  to  be  treated  with  saliva  and  the  precipitation  with 
alcohol  repeated  (Hammarsten). 


CHAPTER  VI. 
SALIVA  AND  SALIVARY  DIGESTION. 

I.  Conduct  of  Saliva  towards  Reagents. 
II.  Detection  of  Mucin. 

III.  Detection  of  Potassium  Sulphocyanate. 

IV.  Detection  of  Ptyalin. 

v.-  Isolation  of  the  Products  of  Salivary  Digestion. 

I.  Conduct  of  Saliva  towards  reagents.* 

1.  The  addition  of  acetic  acid  causes  a  precipitate  insolu- 
ble in  an  excess  of  the  acid  due  to  the  presence  of  mucin. 

2.  Addition  of  nitric  acid:  flocculent  precipitate,  on  heat- 
ing yellow  color;  on  the  addition  of  an  excess  of  caustic  soda 
solution  the  yellow  color  becomes  more  intense  or  changes  to 
orange.     The  reaction  is  due  to  mucin  (and  albumin?). 

3.  Shake  a  little  saliva  with  an  equal  volume  of  water  and 
some  Millon's  reagent:  white  precipitate,  which  gradually 
turns  red  on  boiUng  (mucin). 

4.  Add  caustic  soda  solution  and  then  a  very  small  quan- 
tity of  a  dilute  copper  sulphate  solution:  violet  color  in  con- 
sequence of  the  presence  of  mucin. 

II.  Detection  of  Mucin. 

Twenty  cubic  centimeters  of  saliva  are  poured  into  100  cc. 
of  absolute  alcohol  and  thoroughly  stirred.  The  white  floc- 
culent precipitate  is  filtered  off,  washed  with  alcohol,  then 

*  Use  small  quantities  for  the  tests. 

70 


SALIVA   AND  SALIVARY  DIGESTION.  71 

once  ■^ith  ether,  and  the  filter  placed  in  a  desiccator  for 
twenty-four  hours. 

1.  A  small  portion  of  the  chalky-white  substance  is  treated 
with  water:  it  swells  up  and  becomes  glassy  without  dis- 
solving; on  the  addition  of  a  drop  of  caustic  soda  solution  it 
gradually  dissolves.  This  solution  gives  the  biuret  reaction 
with  caustic  soda  and  a  httle  copper  sulphate  solution. 

2.  Boil  for  a  few  minutes,  in  a  test-tube  with  dilute  hydro- 
chloric acid  (one  part  hydrochloric  acid  to  two  to  three  parts 
of  water),  the  greater  part  of  the  substance  obtained,  cool, 
then  make  alkaline  with  caustic  soda,  add  a  little  copper  sul- 
phate solution  and  heat  to  boiling:  precipitation  of  cuprous 
oxide,  which  is  more  noticeable  when  the  tube  is  cooled  in 
water.  The  reducing  substance  spUt  off  by  boiling  the  mucin 
with  hydrochloric  acid  is  not  sugar  ^  (mucose  of  Fr.  Miiller). 

III.   DETECTION  OF  POTASSIUM    SULPHOCYANATE. 

Add  one  drop  of  hydrochloric  acid  to  a  small  quantity  of 
the  saUva,  then  a  few  drops  of  a  very  dilute  solution  of  ferric 
chloride,  and  shake  thoroughly :  red  coloration  in  consequence 
of  the  formation  of  soluble  ferric  sulphocyanate,  Fe(SCN)3. 

IV.    DETECTION  OF  PTYALIN. 

One  gram  of  starch  is  made  into  a  paste  with  100  cc.  of 
water  (for  details  of  the  method  see  the  chapter  on  Pancreas, 
page  76). 

1.  After  it  has  cooled  to  about  40°  add  to  about  10  cc.  of 
the  paste  approximately  1  cc.  of  saliva,  and  shake  thoroughly. 
The  mixture  becomes  clearer  and  thinner  after  a  few  minutes. 
To  a  part  of  the  solution  obtained  add  a  drop  of  iodine  solu- 
tion (iodine  dissolved  in  an  aqueous  solution  of  potassium 
iodide):  no  blue  coloration,  but  either  a  red  color  (due  to 
erythrodextrin)  or  merely  a  yellow  color  from  the  iodine. 

»  It  is  chitosamine,  see  Zeitschr.  f.  Biolog.  42,  468  (1901).— O. 


72    PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

With  the  other  half  of  the  solution  try  the  Trommer's 
sugar  test.  As  a  check  repeat  the  same  experiment  with 
boiled  saliva:  the  fluid  does  not  become  clear,  the  starch 
remains  unchanged. 

2.  Repeat  experiment  1,  but  digest  for  an  hour  at  40°,  then 
shake  the  solution  obtained  with  some  yeast,  fill  a  fermenta- 
tion-tube with  it,  and  let  stand  at  about  35° :  the  sugar,  mal- 
tose, formed  in  the  salivary  digestion  is  fermentable.^ 

Influence  of  Acids  on  the  Diastatic  Action  of  Ptyalin. 

(a)  Shake  together  thoroughly  10  cc.  of  starch  paste  and 
1  cc.  of  0.27  per  cent,  hydrochloric  acid  (6  cc.  of  hydrochloric 
acid,  1.19  sp.  gr.,  diluted  to  one  hter),  add  1  cc.  of  saHva,  and 
digest  on  the  water-bath  at  40°.  The  diastatic  action  of  the 
enzyme  is  completely  prevented  by  the  acid. 

(&)  Repeat  the  experiment  with  1  cc.  of  dilute  acetic  acid 
of  0.5  to  1  per  cent.  (1  to  2  cc.  of  glacial  acetic  acid  to  200  cc. 
of  water) :  the  diastatic  action  is  not  prevented,  but  it  is  per- 
ceptibly retarded.  An  excellent  arrangement  of  these  ex- 
periments is  also  the  following:^ 

The  mixtures  of  starch  paste  and  saUva  and  in  the  given 
case  also  the  acid  are  placed  in  test-tubes  in  a  water-bath 
whose  temperature  is  kept  at  40°  to  42°.  Each  test-tube  has 
a  pipette  standing  in  it.  From  time  to  time  a  drop  of  the 
mixture  is  taken  out  and  brought  into  contact  with  a  dilute 
solution  of  iodine  in  a  solution  of  potassium  iodide.  For  this 
purpose  place  beforehand  a  number  of  drops  of  the  iodine 
solution  at  regular  intervals  (in  rows)  on  a  porcelain  plate. 
If  we  proceed  in  this  manner,  the  addition  of  a  drop  of  the 
mixture  to  the  iodine  solution  requires  only  the  smallest 
amount  of   time,  so   that  the  time  interval  which  elapses 

^  The  yeast  contains  maltase,  which  inverts  the  maltose.     The  glucose 
formed  then  ferments  with  the  yeast. — O. 
2  Virchow's  Arch.  120,  343. 


SALIVA   AND  SALIVARY  DIGESTION.  73 

between  the  taking  out  of  drops  from  each  of  the  three  mix- 
tures may  be  disregarded,  if  we  work  quickly. 

In  order  to  obtain  a  better  and  clearer  insight  into  the 
progress  of  the  process,  select  for  each  new  test  of  the  mix- 
tures a  new  row  of  drops  of  the  iodine  solution  on  the  plate. 
This  arrangement  enables  us  even  to  recognize  erythrodextrin, 
if  it  is  present  in  sufficient  quantity,  alongside  of  the  starch. 
In  this  case  besides  the  blue  color  due  to  starch  the  red  color 
due  to  erythrodextrin  will  appear  when  the  drops  begin  to 
dry.  Test  the  portions  taken  out  later  also  for  sugar ;  a  very 
small  quantity  is  sufficient  for  this. 

V.  Isolation  of  the  Products  of  Salivary  Digestion. 

Make  25  g.  of  starch  into  paste  with  one  liter  of  water, 
stirring  constantly.  After  cooling  to  40°  (in  order  to  deter- 
mine the  temperature,  the  thick  paste  must  be  well  stirred, 
as  the  distribution  of  the  temperature  in  it  is  very  unequal), 
add  25  cc.  of  saUva,  and  stir  thoroughly  (it  is  advisable  to  use 
saliva  that  has  been  collected  the  previous  day,  as  its  activity 
appears  to  be  greater).  The  paste  very  soon  Uquefies.  As 
soon  as  this  has  happened  pour  the  paste  into  a  cyhnder  and 
digest  for  two  and  a  half  to  three  hours  at  40°.  After  this 
time,  as  a  rule,  the  starch  will  have  disappeared,  the  solution 
no  longer  gives  the  reaction  for  starch  with  iodine.  An  abso- 
lutely exact  statement  of  the  time  necessary  cannot  be  made, 
as  the  activity  of  the  saliva  is  not  always  the  same.  If  the 
starch  has  not  entirely  disappeared,  the  digestion  must  be 
continued.  It  is  not  advisable  to  carry  the  digestion  to  an 
end  in  the  dish  originally  used,  since  the  formation  of  solid 
masses  of  the  paste  on  the  side  of  the  vessel  is  scarcely  to  be 
avoided,  and  these  resist  the  digestive  action  of  the  saliva  for 
a  long  time.  This  again  might  lead  to  a  false  impression  con- 
cerning the  progress  of  the  digestion.  After  the  digestion 
is  completed  heat  the  solution  on  the  water-bath,  filter  from 


74     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

a  small  quantity  of  material  resembling  cellulose,  which  comes 
from  the  starch,  evaporate  on  the  water-bath  ;o  about  100  cc, 
filter  again,  and  evaporate  to  about  25  cc  Pour  this  sirup 
while  still  hot  into  a  flask  containing  100  cc.  cf  90  per  cent, 
alcohol  heated  to  boihng  on  a  water-bath,  heat  a  little  longer, 
shake  thoroughly,  and  let  stand  till  next  day.  The  precipi- 
tate consists  of  dextrin  with  some  maltose;  the  alcoholic  solu- 
tion contains  principally  maltose  with  a  Httle  dextrin  and 
traces  of  glucose.  A  perfect  separation  is  not  attainable  by 
a  single  precipitation  with  alcohol,  and  also  requires  a  larger 
amount  of  material.  On  the  next  day  pour  off  the  alcohoUc 
solution  and  wash  once  with  alcohol  the  glutinous  precipi- 
tate sticking  to  the  flask. 

{a)  Pour  on  the  residue  in  the  flask  about  50  cc.  of  water, 
heat  to  boiling  (with  constant  shaking  so  that  the  precipitate 
may  not  be  burned  or  heated  too  highly),  and  boil  the 
solution  until  the  odor  of  alcohol  has  completely  disap- 
peared. Let  cool,  dilute  to  100  cc,  and  filter  through  a  dry 
filter. 

A  small  portion  of  this  solution  turns  blue-violet  on  the 
addition  of  a  very  small  quantity  of  iodine  solution,  and  red 
with  a  larger  quantity.  Sometimes  these  colors  with  iodine 
are  not  given  because  achroodextrin  has  been  formed.  The 
solution  gives  the  reactions  for  sugar  very  markedly. 

Thirty  cubic  centimeters  of  the  solution  obtained  are 
diluted  to  150  cc.  Add  10  cc.  of  hydrochloric  acid  to  100  cc. 
of  the  solution  and  mark  this  solution  A.  The  remainder, 
50  cc,  is  used  to  determine  the  rotation.  It  amounts  to, 
say,  6.8  per  cent,  calculated  as  glucose. 

The  solution  A  is  then  heated  to  boiling  and  kept  boiling 
gently  for  twenty  minutes.  Let  cool,  fill  up  to  the  former 
volume  (100  cc),  and  again  determine  the  rotation  (neutral- 
ization of  the  solution  may  be  omitted,  if  we  determine  the 
rotation  rapidly  and  clean  out  the  observation-tube  at  once; 


SALIVA   AND  SALIVARY  DIGESTION.  75 

if  we  wish  to  neutralize,  this  must  of  course  be  done  before 
the  volume  is  diluted  to  100  cc).  The  rotation  will  be  found 
to  have  very  materially  decreased;  it  amounts  to  only  about 
one-third  of  the  former  rotation;  in  the  case  given  to  about 
2.6  per  cent.  The  decrease  in  the  rotation  is  due  to  the  trans- 
formation by  boiling  with  acids  of  the  very  strongly  dextro- 
rotatory dextrin  (and  maltose)  into  the  less  strongly  rotating 
glucose. 

(b)  The  alcoholic  solution,  when  evaporated  on  the  water- 
bath,  yields  a  very  sweet-tasting  sirup,  which  gradually 
dries  up,  containing  maltose  with  a  little  dextrin  and  a  very 
small  quantity  of  glucose.  The  maltose  crystallizes  out  of 
this  sirup  only  with  great  difficulty.  It  crystallizes  more 
readily  if  the  digestion  of  the  starch  paste  with  the  saliva  be 
continued  for  a  longer  time  (twenty-four  hours).  On  heat- 
ing with  acids  the  maltose,  like  the  dextrin,  yields  glucose. 
This  conversion  may  be  shown  as  in  the  case  of  dextrin  by 
the  decrease  in  rotation.  For  this  purpose  dissolve  3  g. 
of  the  residue  in  hot  water,  let  cool,  dilute  to  150  cc,  deter- 
mine the  rotation  of  the  solution,  then  treat  100  cc.  of  this 
solution  just  as  in  the  case  of  dextrin,  and  determine  the  rota- 
tion once  more.  It  amounts  to  not  quite  half  as  much  as 
before  boiling  with  the  acid. 

Maltose,  C12H22O11+H2O,  is  formed  together  with  dextrin 
by  the  action  of  malt  (germinated  barley)  on  starch  by  virtue 
of  a  ferment  (diastase)  contained  in  the  malt.  Maltose  crys- 
tallizes in  fine  needles,  it  is  strongly  dextrorotatory  (specific 
rotation  139.2°),  reduces  Fehling's  solution,  is  fermentable,^ 
and  is  found  in  beer.  To  show  the  perfect  fermentability, 
add  to  about  50  cc.  of  a  2  per  cent,  solution  some  yeast,  let 
stand  twenty-four  hours  at  about  35°,  filter,  and  test  the  fil- 
trate for  sugar  by  means  of  Trommer's  test:  no  precipitate 
of  cuprous  oxide  is  formed. 

'  According  to  E.  Fischer,  maltose  is  first  converted  into  glucose  by 
the  maltase  of  the  yeast  and  this  then  ferments. — O. 


CHAPTER  VII. 

EXAMINATION  OF  THE  PANCREAS. 

I.  Tryptic  Digestion. 
II.  Diastatic  Action. 

III.  Cleavage  of  Fats. 

IV.  The  Nucleoproteid  of  the  Pancreas. 

I.  Tryptic  digestion. 

Digest  250  g.  of  fibrin  with  1  liter  of  alkaline  chloroform-water  and  2 
to  2.5  g.  of  pancreas-powder  for  48  hours,  add  acetic  acid,  boil,  and  filter. 


Residue :   coagulated  albumin.  Filtrate    (A)   (reaction  with  bro- 

mine), evaporate,  and  let  stand. 


Crystals  (B)  :  tyrosine.  Filtrate    (C)    evaporate    further: 

leucine,  peptone. 

The  best  material  is  fresh  fibrin.  If  only  the  coagulated  material 
preserved  in  chloroform-water  is  available,  it  is  advisable  to  heat  it 
with  acidified  water  (3  cc.  of  hydrochloric  acid,  specific  gravity  1.19, 
to  1  liter  of  water),  and  then  wash  it  thoroughly  on  a  muslin  filter. 
Sometimes  this  treatment  is  omitted.  The  digestion  takes  place  with 
the  unswollen  fibrin  also,  but  not  so  well.  The  chloroform-water  is 
prepared  by  shaking  1  liter  of  water  with  5  cc.  of  chloroform.  The 
chloroform  is  used  to  prevent  putrefaction:  thymol  may  also  be  used 
for  this  purpose.  If  antiseptic  material  is  not  used,  very  marked  pu- 
trefaction is  certain  to  result.  The  pancreas-powder  is  prepared 
according  to  the  method  of  Kiihne.  Pancreas^  which  has  lain  for 
twenty-four  hours  is  carefully  freed  from  all  visible  fat,  ground  with 
absolute  alcohol,  filtered  after  standing  a  short  time  and  pressed, 

'  From  the  ox  or  dog. 

76 


EXAMINATION  OF  THE  PANCREAS.  77 

ground  with  ether,  again  filtered  and  pressed,  dried  in  the  air  by 
allo^ving  tlie  ether  to  evaporate;  again  ground  and  sifted  through 
wire  gauze,  using  only  the  powder  that  passes  through  the  sieve.  In- 
stead of  using  the  powder  directly  we  may  also  digest  it  for  a  day  at 
40°  v.ith.  chloroform-water  (2.5  g.  :  100  cc),  to  which  some  drops  of 
sodium  carbonate  solution  have  been  added,  and  use  the  filtrate  for 
the  experiment.  The  action  is  usually  somewhat  weaker;  in  many 
cases,  however,  such  a  solution  is  to  be  preferred.  Kiihne  prepares 
an  extract  from  the  pancreas-powder  by  digesting  for  three  or  four 
hours  one  part  of  the  powder  with  five  to  ten  parts  of  a  salicylic  acid 
solution  (0.1  per  cent.),  and  then  filtering. 

The  mixture  of  fibrin,  pancreas-powder,  and  chloroform-water 
must  have  a  distinctly  alkaline  reaction.  When  fresh  fibrin  is  used 
the  addition  of  5  cc.  of  a  concentrated  sodiimi  carbonate  solution  is 
sufficient.  When  coagulated  fibrin  which  has  been  previously  treated 
with  dilute  hydrochloric  acid  is  used  it  may  easily  retain  some  of  the 
acid,  and  in  this  case  the  5  cc.  of  sodium  carbonate  is  insufficient.  We 
must  then  repeatedly  add  sodium  carbonate  solution,  until  even  after 
standing  for  a  considerable  time  the  mixture  has  a  distinctly  alkaline 
reaction. 

The  mixture  is  digested  at  40°  in  a  glass-stoppered  bottle 
for  forty-eight  to  seventy-two  hours  and  should  be  repeatedly 
shaken  thoroughly  during  the  digestion.  After  the  digestion 
make  the  contents  of  the  bottle  quite  faintly  acid  with  acetic 
acid,  heat  to  boiling  in  an  enamelled-iron  dish  or  tinned  ves- 
sel, and  filter, 

1.  To  a  small  portion  of  this  filtrate  (A)  add,  drop  by  drop^ 
and  with  constant  shaking,  some  bromine-water:  the  fluid 
takes  on  a  violet  color:  tryptophan  reaction.  The  rest  of  the 
filtrate  is  evaporated  to  a  thin  sirup  (about  100  cc.)  and  let 
stand  for  some  days  in  a  cool  place.  A  considerable  quantity 
of  white  granular  crystals  of  tyrosine  separates  (B).  Decant 
through  muslin,  put  the  tyrosine  into  a  dish  or  beaker  and 
wash  it  quickly  a  few  times  by  decantation,  then  put  it  into 
a  flask  and  heat  with  water  to  which  a  little  ammonia  has 
been  added,  and  filter.     The  hot  filtrate  is  evaporated  on  the 


78     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

water-bath  until  the  ammonia  has  disappeared;  on  cooling 
the  tyrosine  separates  as  a  chalky  mass;  this  is  filtered  off, 
washed,  and  dried  on  filter-paper. 

Tyrosine,  CgHnNOa,  p-oxyphenyl  a-amino-propionic  acid, 

C6H4  <  ^jj  CHCNH  )COOH  (4)  ^^  ^  constant  cleavage  product 
of  albumin  and  horn  substance  (but  not  of  gelatin  or  of  tissues 
which  may  be  converted  into  gelatin),  formed  by  the  action 
of  dilute  acids  or  alkalies  and  also  on  putrefaction.  It  forms 
shining  silky  needles,  melting  at  310-314°  with  decomposi- 
tion. It  is  very  difficultly  soluble  in  cold  water,  slightly  in 
hot  water,  insoluble  in  alcohol  and  ether,  soluble  in  ammonia 
and  caustic  alkalies. 

Reactions  of  Tyrosine. 

(a)  Heat  a  small  portion  in  a  test-tube  with  some  water 
to  boiling,  let  cool  slowly  (without  cooling  in  water),  and 
examine  the  crystals  under  the  microscope :  tufts  of  needles, 
usually  of  very  regular  form,  which  readily  dissolve  on  the 
addition  of  hydrochloric  acid  and  do  not  melt  when  warmed 
gently  on  the  slide  of  the  microscope  (distinction  from  the 
needles  of  fatty  acids  in  old  pus,  etc.,  which  often  resemble 
tyrosine  needles,  and  from  the  needles  of  fat,  which  form  oily 
drops  on  warming  and  do  not  dissolve  in  hydrochloric  acid). 

(6)  Suspend  a  small  quantity  of  tyrosine  in  water  in  a  test- 
tube,  add  a  few  drops  of  Millon's  reagent  and  heat  gently  till 
boiling  begins.  The  mixture  at  first  turns  rose-red,  then 
gradually  deep  red,  frequently,  however,  not  till  it  has  stood 
some  time ;  if  the  quantity  of  the  tyrosine  is  somewhat  large, 
the  solution  becomes  cloudy  and  gradually  deposits  a  red 
precipitate.  Strong  boiling  is  not  advisable,  as  the  reaction 
is  then  often  not  so  good  and  the  color  is  more  brownish.  All 
benzene  derivatives  in  which  a  hydrogen  atom  of  the  benzene 
nucleus  is  replaced  by  a  hydroxyl  group  give  the  same  reaction. 


EXAMIXATION   OF   THE  PANCREAS.  79 

(c)  Piria's  Test,  due  to  the  formation  of  tyrosine  sulphoiiic 
acid.  Pour  on  a  little  tyrosine  in  a  dry  test-tube  some  con- 
centrated sulphuric  acid,  place  the  tube  in  an  actively  boihng 


Fig.  5. — Leucine  and  Tyrosine. 

water-bath,  and  leave  it  there  for  about  half  an  hour.  Let 
cool,  pour  into  several  times  its  volume  of  water,  rinse  with 
water,  and  grind  the  solution,  diluting,  if  necessary,  wdth 
barium  carbonate,  added  in  portions,  until  the  solution  no 
longer  reacts  acid.  Filter,  evaporate  to  a  few  cubic  centi- 
meters, and  add  cautiously  some  very  dilute  ferric  chloride 
solution:   violet  color. 

(d)  Reaction  of  Deniges.  Gently  heat  a  small  portion  ol 
the  tyrosine  with  2  to  3  cc.  of  concentrated  sulphuric  acid  to 
which  a  few  drops  of  formalin  have  been  added:  brownish- 
red  color,  which  turns  green  on  the  addition  of  glacial  acetic 
acid.  Neither  albumin  nor  peptone  reacts  with  this  reagent 
of  Deniges.^ 

*  According  to  C.  T.  Momer  the  reagent  is  made  as  follows* 
1  volume  formalin; 
45  volumes  di.stilled  water; 
55  volumes  concentrated  sulphuric  acid. 
If  a  portion  of  this  solution  (2  cc.J  is  tnsated  with  a  little  tyrosine  (in  the 
solid  form  or  in  solution)  and  the  inixture  is  heated  to  boiling,  a  beautiful 
preen  color  appears.     Zeitschr.  f.  physiol.  Cheni.  37,  86. — O. 


80     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

2.  The  solution  C  decanted  from  the  tyrosine  is  evaporated 
further  on  the  water-bath:  crystals  of  leucine  form  on  the 
surface.  These  are  to  be  examined  under  the  microscope: 
weakly  refracting  balls  or  aggregates,  sometimes  with  a  recog- 
nizable racUating  structure,  which  dissolve  readily  in  hydro- 
chloric acid  and  caustic  alkalies. 

The  solution  is  evaporated  to  a  sirup,  treated  with  several 
times  its  volume  of  90  per  cent,  alcohol,  put  into  a  flask, 
heated  on  the  water-bath  to  boiling,  and,  after  it  is  perfectly 
cold,  filtered.  The  alcoholic  extract  contains  a  good  deal  of 
leucine  besides  a  little  peptone;  the  insoluble  residue,  much 
peptone  with  a  small  quantity  of  leucine.  The  alcoholic 
extract  is  evaporated  to  dryness,  the  residue  dissolved  in 
water,  the  solution,  boiled  with  lead  hydroxide,  and,  after 
coohng,  filtered.  The  filtrate  freed  from  lead  by  means  of 
hydrogen  sulphide  is  filtered,  evaporated  to  a  small  volume, 
and  the  leucine,  which  separates  on  standing,  dried  on  a  clay 
plate. 

Leucine,  a-amino-isobutyl-acetic  acid,  (CH3)2CHCH2- 
CH(NH2)C00H,  is  a  constant  cleavage  product  of  albumin, 
horn  substance,  gelatin,  and  tissues  yielding  gelatin,  formed 
by  the  action  of  dilute  acids  or  alkahes  and  also  on  putrefac- 
tion. It  forms  in  pure  condition  shining  white  leaflets  which 
are  wet  by  water  only  with  difficultly.  It  dissolves  in  40  to 
46  parts  of  cold  water,  more  readily  in  hot,  and  with  diffi- 
culty in  alcohol,  but  is  very  much  more  readily  soluble  in 
these  solvents  in  the  impure  condition. 

Reactions  of  Leucine. 

(a)  A  little  of  the  substance,  when  heated  cautiously  in 
an  open  tube  held  slantingly,  forms  a  woolly  subUmate  of 
leucine;  an  odor  of  amylamine  is  evolved  at  the  same  time, 
a  part  of  the  leucine  undergoing  decomposition. 

(6)  Put  a  piece  of  caustic  potash  (stick  form)  about  1  cm. 


EXAMINATION  OF  THE  PANCREAS.  81 

long  into  a  test-tube  with  a  little  leucine  and  one  to  two  drops 
of  water.  Heat  till  the  alkali  melts,  when  a  strong  evolu- 
tion of  ammonia  takes  place.  Let  cool,  dissolve  the  melted 
mass  in  a  httle  water,  and  acidify  with  dilute  sulphuric  acid: 
odor  of  valeric  acid.  Leucine  by  this  treatment  takes  up 
oxygen  and  is  decomposed  into  ammonia,  carbon  dioxide, 
and  valeric  acid. 

(c)  Dissolve  some  leucine  in  water,  decolorize  the  solution, 
if  necessary,  with  some  good  bone-black,  filter,  make  alkaline 
Tvith  caustic  soda  solution,  and  then  add  one  to  two  drops 
of  copper  sulphate  solution :  the  precipitate  of  copper  hy- 
droxide, which  first  forms,  dissolves  to  a  blue  solution  of 
leucine  copper,  which  is  not  reduced  on  heating. 

3.  The  residue  insoluble  in  alcohol,  which  has  been  freed 
more  or  less  completely  from  leucine,  is  treated  with  absolute 
alcohol,  filtered,  and  washed  with  alcohol  and  ether.  Test 
this  for  albumose  and  peptone  by  dissolving  it  in  a  Httle 
water  and  saturating  with  ammonium  sulphate,  etc.  (See 
chapter  on  Gastric  Digestion,  page  33).  Ultimately  the  pep- 
tone itself  is  to  be  isolated. 

II.  DiASTATic  Action  of  the  Pancreas. 

Prepare  some  starch  paste  as  follows:  Measure  off  100  cc. 
of  water,  grind  1  g.  of  starch  (potato-starch)  in  a  mortar  with 
a  part  of  the  water,  pour  the  fluid  into  a  dish,  rinse  the  mortar 
with  the  rest  of  the  100  cc.  of  water,  and  heat  the  mixture  to 
boiling  with  constant  stirring.  Then  treat  1  g.  of  pancreas- 
powder  with  50  cc.  of  water,  digest  for  two  hours  at  40°,  and 
filter.  Mix  in  a  test-tube  equal  volumes  of  the  starch  paste 
and  the  pancreas  extract,  and  digest  at  40°.  The  starch  paste 
liquefies  and  becomes  transparent.  At  this  point  the  solu- 
tion gives  the  reaction  for  sugar  and  no  longer  turns  blue  on 
the  addition  of  iodine,  but  either  turns  red  (erythrodextrin) 
or  gives  no  color  at  all. 


82     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

As  a  check,  repeat  the  same  experiment,  but  boil  the  pan- 
creas extract  before  using  it:  the  saccharifying  action  does' 
not  take  place. 

Instead  of  the  aqueous  pancreas  extract  we  may  use  a  glycerin 
extract  of  the  pancreas  (about  ten  drops  to  10  cc.  of  starch  paste),  or 
the  fresh  pancreas  when  that  is  available.  Grind  a  piece  of  the 
pancreas  (from  the  ox  or  dog)  with  water  to  a  thin  paste,  filter 
through  muslin,  and  mix  about  equal  volumes  of  starch  paste  and 
the  gland  extract. 

III.    DETECTION  OF  THE  LIPOLYTIC  FERMENT,  LIPASE. 

This  succeeds  only  with  the  fresh  pancreas.  Grind  the 
finely  minced  pancreas  to  a  thin  paste,  divide  it  into  two 
equal  parts,  boil  one  part  (A)  to  destroy  the  ferment,  but  not 
the  other  (B).  Then  shake  a  few  grams  of  butter-fat  with 
luke-warm  water,  add  a  few  drops  of  rosoUc  acid  solution 
and  then  tenth-normal  caustic  soda  solution  till  the  mixture 
is  distinctly  red.  Now  mix  equal  parts  of  the  fat  emulsion 
with  the  pancreas-paste,  one  part  with  (A),  the  other  with 
(B),  and  add  a  drop  or  two  of  chloroform.  If  these  mixtures 
are  not  distinctly  red,  then  add  cautiously,  drop  by  drop, 
dilute  sodium  carbonate  solution.  Digest  the  mixture  twelve 
to  twenty-four  hours  at  40°.  The  solution  marked  (A)  does 
not  change  its  color;  (B)  becomes  yellow  in  consequence  of 
the  butyric  acid  set  free  by  the  hydrolysis  of  the  butyrin. 

In  like  manner  we  can  test  cystic  fluids,  which  are  suspected  to 
come  from  the  pancreas,  for  the  saccharifying  and  lipolytic  ferments. 
To  detect  the  presence  of  trypsin  digest  a  portion  of  the  fluid,  made 
alkaline,  if  necessary,  for  twenty-four  hours  at  40°.  Since  albumin  is 
a  constant  constituent  of  the  cystic  fluids,  they  must  now  contain 
peptone  and  tryptophan  in  case  trypsin  is  present.  Coagulate  the 
albmnin  (adding  water  if  necessary)  by  heating  the  solution  faintly 
acidified  with  acetic  acid,  filter,  and  divide  the  filtrate  into  two  parts. 
One  part  is  used  for  the  biuret  reaction  with  caustic  soda  and  copper 
sulphate  solutions,  the  other  for  the  tryptophan  reaction.  To  detect 
very  small  quantities  of  the  digestion  products  of  albumin  we  may 


EXAMINATION  OF   THE  PANCREAS.  83 

also  precipitate  the  filtrate  or  a  part  of  it  ^vith  phosphotungstic  acid, 
after  first  acidifying  strongly  with  hydrocliloric  acid.  Then  warm 
the  tube,  when  the  precipitate  becomes  more  dense,  packs  together, 
and  usually  sticks  to  the  glass.  Rinse  it  a  few  times  with  water,  dis- 
solve in  dilute  caustic  soda  solution,  shake  till  the  blue  color,  which 
forms  at  first,  disappears,  then  cautiously  add  the  copper  sulphate 
solution. 


IV.  NUCLEOPROTEID  OF  THE  PANCREAS. 

The  pancreas,  according  to  Hammarsten,  contains  a  nucleo- 
proteid,  which,  according  to  Bang,  may  be  split  up  into  albu- 
min and  an  acid,  guanylic  acid.  The  latter  yields  on  cleavage 
phosphoric  acid,  guanine,  and  a  carbohydrate  containing  five 
atoms  of  carbon,  a  pentose  of  the  composition  CsHioOg.  The 
nucleoproteid  (nucleoalbumin)  itself  also  yields  the  same 
cleavage  products,  as  Hammarsten  had  already  found  before 
Bang. 

Detection:  Heat  200  g.  of  finely  chopped  pancreas  to 
boihng  with  one  Hter  of  water,  keep  boiUng  for  ten  minutes, 
filter,  and  add  cautiously  to  the  filtrate,  while  still  warm, 
about  10  to  15  cc.  of  30  per  cent,  acetic  acid,  until  a  fine 
flocculent  precipitate  begins  to  settle.  If  the  precipitate  does 
not  settle  well  it  is  advisable  to  heat  again.  Filter,  wash 
with  water,  remove  the  precipitate  from  the  filter,  grind  it 
with  50  cc.  of  absolute  alcohol,  filter,  treat  the  precipitate 
with  about  50  cc.  of  ether,  filter  next  day,  wash  once  with 
ether,  and  grind.  The  presence  of  phosphorus  and  pentose 
may  be  very  easily  shown  in  the  somewhat  impure  nucleo- 
proteid thus  obtained. 

1.  To  detect  the  phosphorus  fuse  a  small  quantity  with 
the  oxidizing  mixture  and  then  proceed  as  directed  under 
Casein,  page  9. 

2.  To  detect  the  pentose  the  phloroglucin  and  the  orcin 
tests  may  be  used. 


84     PHYSIOLOGICAL  AND   PATHOLOGICAL  CHEMISTRY. 

(a)  Phloroglucin  Test.  Pour  on  a  very  small  quantity  of 
the  substance  a  few  cubic  centimeters  of  hydrochloric  acid, 
add  a  Uttle  phloroglucin,  and  heat  to  boihng:  cherry-red 
color,  then  turbidity.  Let  cool  somewhat,  shake  with  an 
equal  volume  of  amyl  alcohol,  and  examine  this  with  the 
spectroscope:   absorption-band  between  D  and  E. 

(6)  Orcin  Test.  Instead  of  phloroglucin  take  a  few  orcin 
crystals  and  proceed  in  the  same  way:  reddish-blue  color, 
then  precipitation  of  a  blue  pigment.  The  amyl  alcohol  turns 
red  and  after  some  time  emerald-green.  Examine  with  the 
spectroscope:   absorption-band  between  C  and  D. 

If  somewhat  larger  quantities  (not  less  than  0.2  g.)  of  the 
nucleoproteid  are  available,  the  presence  of  the  guanine  may 
;also  be  shown.  Heat  in  a  flask,  cautiously  and  shaking  con- 
istantly,  with  about  25  cc.  of  a  mixture  of  one  volume  of  hydro- 
chloric acid  and  three  volumes  of  water.  Keep  boiling  gently 
about  fifteen  minutes,  neutralize  with  caustic  soda  solution, 
acidify  with  acetic  acid,  and  let  stand  till  next  day:  guanine 
will  separate.  In  the  filtrate  the  phosphoric  acid  may  be 
detected  by  means  of  uranium  acetate,  the  pentose  by  means 
of  the  Trommer's  test  and  the  pentose  tests  given  above. 


CHAPTER  VIII. 

EXAMINATION  OF  BILE. 

I.  Detection  of  the  Constituents  of  Bile. 
II.  Detection  of  Mucin  in  Bile. 
III.  Preparation  of  Taurine. 

I.   DETECTION  OF   THE   CONSTITUENTS   OF  BiLE. 

Mix  200  cc.  of  ox-bile  with  bone-black,  evaporate  to  dryness,  heat  with 
absolute  alcohol,  and  filter. 


Residue :  mucin,  salts,  and  Alcoholic  solution  add  ether, 

bone-black. 


1 1 

Precipitate :  salts  of  the  bile-acids.  Solution  contains  cholesterin. 

Two  hundred  cubic  centimeters  of  ox-bile  are  evaporated 
as  nearly  to  dryness  as  possible  on  the  water-bath  with  some 
good  bone-black  (one-fourth  the  volume),  the  residue  removed 
from  the  dish  after  cooling,  put  into  a  flask,  and  extracted  on 
the  water-bath  with  alcohol.  After  cooling,  filter,  evaporate 
the  extract  to  dryness  on  the  water-bath,  dissolve  the  dry 
mass  in  absolute  alcohol,  and  filter  into  a  dry  flask.  Add  to 
the  filtrate  anhydrous  ether  until  the  cloudiness  becomes, 
permanent.  After  long  standing,  sometimes  even  on  the  next 
clay,  a  mixture  of  the  sodium  salts  of  glycocholic  acid, 
C^H^jNOe,  and  taurocholic  acid,  CjeH.sNSOj,  crvstallizes 
(Plattner's  crystallized  bile). 

The  bile-acids  are  distinguished  by  a  reaction  which  is 

85 


86     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

also  given  by  cholic  acid  (cholalic  acid),  a  cleavage  product 
of  both  bile-acids. 

Pettenkofer's  Test  for  the  Bile-acids. 

Use  either  a  1  per  cent,  solution  of  the  crystallized  salts 
of  the  bile-acids  or  of  the  commercial  fel  tauri  depurat.  sice. 

Add  to  a  few  cubic  centimeters  in  a  test-tube  five  drops  of 
a  10  per  cent,  solution  of  cane-sugar  (or  add  a  small  piece  of 
cane-sugar  and  dissolve  this  by  shaking) .  Then  let  about  half 
the  volume  of  concentrated  sulphuric  acid  flow  slowly  down 
the  side  of  the  tube,  held  in  a  slanting  position,  so  that  the  sul- 
phuric acid  forms  the  under  layer.  At  the  surface  of  contact  of 
the  two  liquids  a  purple-violet  color  appears.  Dip  the  test- 
tube  into  a  cylinder  or  beaker  filled  with  water  and  mix  the 
sulphuric  acid  and  the  solution  of  the  bile-acids,  but  not  too 
quickly,^  by  moving  the  test-tube  around  the  walls  of  the 
vessel  in  circles;  a  deep  purple  solution  results.  For  exam- 
ination with  the  spectroscope  pour  a  little  of  the  solution 
into  a  few  cubic  centimeters  of  glacial  acetic  acid  and  about 
the  same  amount  into  some  alcohol. 

(a)  The  acetic  acid  solution  shows  an  absorption-band  in 
the  green  and  a  more  or  less  pronounced  greenish  fluorescence. 

(6)  The  alcoholic  solution  also  shows  immediately  after 
mixing  only  this  one  band,  but  very  soon  it  takes  on  a  brown- 
ish shade  and  then  shows  two  pronounced  absorption-bands 
in  the  green  and  in  the  blue. 

Modification  of  the  Test  according  to  Mylius.^ 

Instead  of  the  cane-sugar  use  a  drop  of  furfurol  solution 
(a  drop  of  furfurol  shaken  thoroughly  in  a  test-tube  with 
10    c.c.    of   water).     The   reaction   develops   more   slowly, 

^  If  the  temperature  exceeds  70°  during  the  mixing  the  pigment  is 
destroyed. 

^  Zeit.  fiir  physiol.  Chemie,  11,  493. 


EXAMINATION  OF  BILE.  87 

requires  apparently  more  sulphuric  acid,  and  is  often  not 
equal  in  intensity  to  the  origmal  Pettenkofer's  reaction. 

According  to  v.  Udranszky  ^  the  best  proportions  for  this 
reaction  are :  1  cc.  of  the  alcoholic  solution  of  the  bile-acids, 
one  drop  of  the  furfurol  solution  (0.1  per  cent.),  and  1  cc.  of 
concentrated  sulphuric  acid. 

Modification  according  to  Neukomm. 

Use  a  0.1  per  cent,  solution  of  the  salts  of  the  bile- 
acids.  Add  to  some  drops  of  this  solution  a  trace  of  sugar 
solution,  then  one  or  more  drops  of  dilute  sulphuric  acid, 
and  evaporate  in  a  dish  on  the  water-bath:  a  violet  color 
develops  at  the  edge  of  the  evaporating  mixture.  As  soon 
as  this  is  distinctly  perceptible  stop  the  evaporation. 

Preparation  of  Glycocholic  Acid. 

Dissolve  the  rest  of  the  crystallized  bile  (with  the  excep- 
tion of  a  small  part  to  be  kept)  in  a  little  water,  pour  some 
ether  on  top  of  this  solution,  and  then  add  dilute  sulphuric 
acid  until  a  permanent  and  marked  cloudiness  appears.  The 
glycocholic  acid  separates  gradually  in  fine  silky  needles. 
Glycocholic  acid  is  very  difficultly  soluble  in  cold  water, 
more  readily  in  hot,  and  easily  soluble  in  alcohol.  The  aque- 
ous solution  has  a  bitter-sweet  taste,  reacts  acid,  and  decom- 
poses the  alkaline  carbonates  when  heated  with  them.  The 
acid  as  well  as  the  salts  are  dextrorotatory. 

Preparation  of  Taurocholic  Acid. 

The  preparation  of  this  acid  is  best  accomplished  by 
using  dog's  bile,  in  which  it  is  the  only  acid  present.  To 
show  its  presence  in  the  crystallized  bile,  fuse  0.2  g.  of  this 

»  Zeit.  fiir  physiol.  Chemie,  12,  371. 


88     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

with  6  g.  of  the  oxidizing  mixture  and  test  for  sulphuric  acid 
in  the  fused  mass  (see  chapter  on  Milk,  page  7). 

Detection  of  Cholesterin. 

Cholesterin  is  present  in  the  alcoholic-ethereal  fluid, 
together  with  the  salts  of  the  bile-acids  stiU  remaining  in  solu- 
tion. Allow  the  greater  part  of  the  ether  to  evaporate  by- 
letting  the  alcoholic-ethereal  solution  stand  in  an  open  dish, 
remove  the  rest  of  the  ether  and  alcohol  by  evaporation  on 
the  water-bath,  dissolve  the  residue  in  water,  shake  the  mix- 
ture with  ether,  remove  the  ether  and  evaporate  the  ether 
extract.  Try  the  cholesterin  reaction  with  the  residue  (see 
Chapter  IX,  Examination  of  Biliary  Calculi,  page  90). 

II.  MUCIN  OF  THE  Bile. 

On  the  addition  of  acetic  acid  to  100  cc.  of  bile  there  is 
formed  a  resinous  precipitate,  which  is  ordinarily  called  bile- 
mucin,  but  whose  exact  nature  is  still  doubtful.  The  pre- 
cipitate also  contains  glycocholic  acid,  which  may  be  removed 
by  extraction  with  alcohol. 

III.   PREPARATION  OF   TAURINE. 

Heat  300  cc.  of  bile  in  an  evaporating-dish  on  the  sand- 
bath  with  100  cc.  of  hydrochloric  acid  until  the  resinous 
mass,  which  separates  at  first,  the  so-called  choloidic  acid,  is 
converted  into  dyslysin  (anhydride  of  cholic  acid).  This 
point  may  be  determined  by  drawing  out  the  resinous  mass 
in  threads  with  a  glass  rod.  These  should  sohdify  at  once 
and  should  then  be  quite  brittle.  It  may  then  be  assumed 
that  all  the  taurocholic  acid  is  decomposed.  Decant  from 
the  dyslysin  and  evaporate  until  sodium  chloride  begins  to 
separate,  filter,  evaporate  on  the  water-bath  to  a  small  volume 
(the  salt  which  separates  is  to  be  removed  by  filtering  again), 
pour  the  fluid  remaining  into  about  fifteen  times  its  volume 


EXAMINATION  OF  BILE.  89 

of  alcohol  (or  mix  \vith  it),  after  twenty-four  hours  filter 
off  the  taurine  which  has  separated,  wash  with  alcohol 
and  recrystallize  it  from  hot  water,  using  some  bone- 
black  to  decolorize.  Taurine  (amino-ethyl  sulphonic  acid), 
CH2NH2 

I  ,  crystallizes  in  large,  transparent,  glittering  prisms, 

CH2SO3H 

which  are  readily  soluble  in  hot  water,  more  difficultly  in 
cold,  and  insoluble  in  absolute  alcohol. 

1.  Heat  a  few  crystals  on  platinum-foil:  the  taurine 
melts,  turns  brown  and  carbonizes  on  heating  more  strongly, 
developing  a  suffocating  odor  (sulphurous  acid  [and  sulphuric 
acid  ?]). 

2.  Powder  a  few  crystals,  mix  with  several  times  their 
volume  of  dry  sodium  carbonate,  and  fuse  on  platinum-foil. 
After  cooling,  dissolve  the  fused  mass  in  water,  put  it  into 
a  test-tube,  and  add  some  dilute  sulphuric  acid:  odor  of 
hydrogen  sulphide.  Moisten  a  strip  of  filter-paper  with  a 
solution  of  lead  acetate  and  remove  the  excess  of  the  solu- 
tion by  pressing  between  filter-paper.  When  held  over  the 
opening  of  the  test-tube  this  paper  turns  brown  or  black 
owing  to  the  formation  of  lead  sulphide. 


CHAPTER  IX. 
EXAMINATION  OF  BILIARY  CALCULI. 

Powdered  gall-stones  extracted  with  ether  in  a  flask  and  filtered. 


Solution  evaporated :  cholesterin  (A).     Residue  (B)  treated  on  the  filter 

with  dilute  hydrochloric  acid. 


Solution  (C)  calcium  salts,  some-  Residue   (D)  washed  with  water, 

times  traces  of  copper.  dried  and  extracted  with  chlo- 

roform :      bile-pigments,     espe- 
cially bilirubin. 

Pour  on  the  finely  powdered  gall-stones  ^  (about  2  g.)  in 
a  dry  flask  about  ten  times  the  volume  of  ether,  shake  a  few 
times,  then  filter  through  a  dry  filter,  and  evaporate  the 
ethereal  solution. 

1.  The  cholesterin  thus  obtained,  C27H43OH,  is  not  quite 
pure,  but  often  contains  some  fat.  It  is  sufficiently  pure, 
however,  for  the  following  reaction:  ^ 

(a)  Dissolve  a  part  of  the  cholesterin  in  hot  alcohol,  let 
the  solution  evaporate  spontaneously  on  a  watch-glass,  and 
examine  the  mass  of  cholesterin  crystals  resulting  under  the 
microscope:    rhombic   tablets,   frequently  with  re-entering 

1  Mixture  of  cholesterin  and  pigment-stones. 

^  To  purify  it,  dissolve  in  80  per  cent,  alcohol,  add  a  piece  of  caustic 
potash,  warm  in  a  flask,  on  the  water-bath,  evaporate  to  drjmess  in  a  dish 
on  the  water-bath,  take  up  the  residue  with  water,  shake  the  mixture  with 
ether  (free  from  alcohol),  remove,  and  evaporate  the  ethereal  extract. 

90 


EXAMIXATION  OF  BILIARY   CALCULI.  91 

angles.  These  appear  beautifully  formed  when  they  have 
separated  spontaneously  from  old  exudates,  transudates,  or 
cystic  fluids. 

(b)  Place  a  small  quantity  of  the  cholesterin  crystals  on 
a  shde,  put  on  the  cover-glass,  and  let  a  drop  of  a  mixture  of 


Fig.  6. — Cholesterin  from  Cystic  Fluid. 

five  volumes  of  concentrated  sulphuiic  acid  and  one  volume 
of  water  flow  under  the  cover-glass  from  the  side  and  then  a 
very  small  quantity  of  a  solution  of  iodine;  the  cholesterin 
crystals  gradually  turn  brown  or  violet,  sometimes  even  a 
clear  blue,  and  partially  dissolve.  The  coloring  never  appears 
entirely  uniform  and  is  often  incomplete. 

(c)  Use  the  greater  part  of  the  cholesterin  for  the  follow- 
ing reactions: 

1.  Evaporate  a  small  quantity  on  the  cover  of  a  porcelain 
crucible  with  hydrochloric  acid  and  a  trace  of  ferric  chloride: 
blue  color. 

2.  Chloroform  Sulphuric  Acid  Reaction.  Dissolve  some 
cholesterin  in  a  dry  tcst-tulx;  in  a  few  cul)ic  centimeters  of 
chloroform,  add  an  equal  volume  of  concentrated  sulphuric 


92     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

acid,  and  shake  thoroughly  several  times.  The  chloroform 
solution  turns  bloocl-red,  gradually  changes  to  cherry-red, 
and  finally  to  purple.  The  sulphuric  acid  under  the  chloro- 
form solution  shows  a  greenish  fluorescence.  If  now  some  of 
the  chloroform  solution  is  poured  into  a  wet  test-tube  and 
shaken,  it  will  quickly  become  colorless,  and  upon  adding 
sulphuric  acid  the  original  red  color  will  be  restored.  When 
poured  out  into  a  dish  the  chloroform  will  also  become  color- 
less from  the  moisture  which  it  abstracts  from  the  air.  K 
the  purple-colored  chloroform  solution  is  diluted  by  the  addi- 
tion of  more  chloroform,  it  often  turns  blue  (in  consequence 
of  a  small  amount  of  water  in  the  chloroform),  and  the  addi- 
tion of  sulphuric  acid  turns  it  red  again.  In  very  dilute 
solutions  (a  trace  of  cholesterin  dissolved  in  chloroform)  the 
reaction  is  somewhat  different,  but  also  characteristic:  yel- 
low to  rose  color  of  the  chloroform,  yellow  color  of  the  sul- 
phuric acid  with  a  greenish  fluorescence. 

3,  Liebermann-Burchard  Reaction.  Dissolve  a  small  quan- 
tity of  the  cholesterin  in  a  few  cubic  centimeters  of  chloroform 
in  a  dry  test-tube,  add  two  or  three  drops  of  acetic  anhydride, 
and  then  concentrated  sulphuric  acid,  drop  by  drop.  A  rose 
color  first  develops,  then  a  beautiful  blue,  which  finally  turns 
bluish  green.  If  only  a  very  small  quantity  of  cholesterin 
is  used,  the  green  color  develops  after  standing  a  few  min- 
utes. 

Both  tests,  2  and  3,  are  equally  dehcate.  Not  only  cho- 
lesterin but  also  its  esters,  such  as  those  of  palmitic  and 
stearic  acid  occurring  in  nature  (Liebreich's  lanoHn),  give 
the  reactions. 

If  a  somewhat  larger  quantity  of  cholesterin  is  available, 
purify  it  by  repeated  recrystalHzation  from  hot  absolute 
alcohol,  press  the  crystals  between  filter-paper,  and  deter- 
mine the  melting-point.  It  melts  at  145°  (distinction  from 
the  cholesterin  occurring  in  plants,  phytosterin,  which  gives 


EXAMINATION  OF  BILIARY  CALCULI.  93 

a  very  similar  chloroform  sulphuric  acid  reaction,  but  whose 
melting-point  is  133-136°). 

2.  The  residue  B  remaining  on  the  filter  is  washed  a  few 
times  with  ether,  the  upper  part  of  the  filter,  which  still  con- 
tains some  cholesterin,  is  cut  ofT,  the  filter  filled  with  dilute 
hydrochloric  acid  (1  :  3),  and  the  filtrate  poured  repeatedly 
upon  the  residue. 

3.  The  filtered  solution  C  contains  calcium  salts,  which 
may  be  proved  by  making  a  portion  of  the  solution  alkahne 
"with  ammonia,  and  adding  acetic  acid  and  ammonium  ox- 
alate; sometimes  traces  of  copper  are  present,  as  may  be 
shown  by  the  addition  of  a  few  drops  of  potassium  ferro- 
cyanide:  brown  color  or  precipitate  of  copper  ferrocyanide. 

4.  The  residue  D  remaining  on  the  filter  is  washed  with 
water  till  the  wash-water  is  free  from  hydrochloric  acid,  the 
filter  is  then  dried  in  an  air-bath,  cut  into  pieces,  and  these 
are  heated  in  a  dry  flask  with  a  little  chloroform.  The  brown- 
ish-yellow fluid  resulting  is  then  filtered  through  a  dry  filter. 

The  solution  contains  bilirubin,  C32H3gN4,Oe.  Let  a  por- 
tion of  this  solution  evaporate  spontaneously  on  a  watch- 
glass  and  examine  the  residue  under  the  microscope:  small, 
ill-defined,  elongated  rhombic  plates  or  indistinct  crystalline 
grains  of  bilirubin  (not  to  be  confused  with  the  cholesterin 
crystals,  which  may  still  be  present).  Pour  on  the  edge  of 
the  watch-glass  a  drop  of  nitric  acid  which  contains  some 
nitrous  acid:  play  of  colors  of  the  Gmelin  reaction  (see  chap- 
ter on  Urine,  *' Bile-pigments,"  page  121).  Bilirubin^  is 
isomeric  with  hsematoporphyrin  (page  56),  which  gives 
GmeUn's  reaction  indistinctly. 

Shake  up  the  greater  part  of  the  solution  with  a  weak 
solution  of  sodium  hydroxide:  the  pigment  goes  over  into 
the  alkaline  solution,  while  the  chloroform  is  more  or  less 

'  According  to  Zalcski  haematoporphyrin  has  the  formula  Cg^HagN^Og 
and  is  not  isomeric  with  bilirubin,  Zeit.  physiol.  Chem.  37,74  (1902). — O. 


94    PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

completely  decolorized  (distinction  from  lutein,  occurring  in 
the  yolk  of  eggs,  corpus  luteum,  many  cysts,  blood-serum, 
palm-oil,  and  in  flowers,  which  is  not  removed  from  the 
chloroform  solution  by  shaking  with  dilute  alkahes). 

Separate  the  alkahne  solution  and  let  it  stand  in  the  air: 
it  gradually  turns  green  owing  to  the  formation  of  biliverdin, 
C,2H,eN,0«(?)  (oxidation). 


CHAPTER  X. 
EXAMINATION  OF  THE  URINE. 

I.  General  Properties. 

Note  the  color  of  the  urine  and  whether  it  is  clear  or 
cloudy ;  test  the  reaction  of  the  fresh  urine  with  litmus  paper, 
and  determine  the  specific  gravity  with  a  urinometer.  Let 
a  portion  stand  for  some  time,  note  what  happens,  and  again 
determine  the  reaction. 

II.  Conduct  towards  Reagents. 

Treat  small  portions  of  urine  with  the  following  reagents: 

1.  Caustic  Soda:  turbidity,  precipitation  of  the  phos- 
phates of  calcium  and  magnesium,  which,  on  heating,  become 
more  dense  and  settle  to  the  bottom;  they  always  appear 
slightly  colored;  on  standing  crystals  of  ammonium  mag- 
nesium phosphate  are  also  deposited. 

2.  Hydrochloric  Acid :  dark  coloration,  especially  on  warm- 
ing, sometimes  a  distinct  red  coloration;  on  standing  crystal- 
line deposit  of  uric  acid. 

3.  On  boiling,  the  urine  remains,  as  a  rule,  clear  and  its 
reaction  acid;  frequently,  however,  it  becomes  cloudy,  due 
to  the  precipitation  of  calcium  phosphate.  In  this  case  the 
reaction  is  either  neutral  or  alkaline.  This  precipitate  readily 
dissolves  on  the  addition  of  a  few  drops  of  acetic  acid,  while 
that  due  to  albumin  (which  resembles  this  phosphate  precipi- 
tate very  closely  in  appearance)  remains  undissolved. 

95 


96     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

4.  Barium  Chloride:  white  precipitate  of  barium  phos- 
phate and  sulphate ;  on  the  addition  of  hydrochloric  acid  the 
barium  phosphate  dissolves  and  the  quantity  of  the  precipi- 
tate diminishes  perceptibly. 

5.  Silver  Nitrate :  white  precipitate  of  silver  chloride  and 
silver  phosphate;  on  the  addition  of  nitric  acid  the  latter 
dissolves,  silver  chloride  remains. 

6.  Basic  Lead  Acetate:  heavy  precipitate,  which  consists 
principally  of  lead  chloride,  lead  phosphate,  and  lead  sul- 
phate, together  with  the  greater  part  of  the  coloring  matter 
of  the  urine.  The  filtrate  is  colorless  or  almost  colorless.  The 
precipitation  with  basic  lead  acetate  is  frequently  used  to 
decolorize  the  urine. 

III.  Preparation  of  urea. 

Two  hundred  to  three  hundred  cubic  centimeters  of  dog's 
urine  or  double  the  quantity  of  human  urine  are  treated  with 
baryta  mixture  (one  volume  of  a  saturated  solution  of  barium 
nitrate  and  two  volumes  of  baryta- water),  until  a  portion  of 
the  urine  when  taken  out  and  filtered  no  longer  gives  a  pre- 
cipitate with  the  mixture ;  filter  off  the  precipitate  of  barium 
phosphate  and  sulphate,  wash  once  with  water  (the  precipi- 
tate, after  washing,  may  be  thrown  away),  and  evaporate 
the  filtrate  ,to  a  sirup,  at  first  over  a  free  flame  and  then  when 
the  volume  amounts  to  about  200  cc.  on  the  water-bath. 
Precipitate  with  about  150  cc.  of  alcohol,  and  after  half  an 
hour  filter  from  the  precipitate,  which  consists  of  salts  and 
extractive  material.  Evaporate  the  filtrate  on  the  water- 
bath  as  nearly  to  dryness  as  possible,  and  after  cooling  add 
double  the  volume  or  somewhat  more  of  nitric  acid  (one  part 
concentrated  nitric  acid  to  one  part  of  water).  The  urea 
nitrate  is  filtered  off  (preferably  next  day),  washed  with  some 
cold  nitric  acid,  drained  thoroughly,  and  dried  on  a  porous  clay 
plate  or  on  filter-paper.     In  order  to  conveH  the  urea  nitrate 


EXAMINATION  OF  THE   URINE.  97 

into  urea,  dissolve  it  in  water  in  a  dish,  add  barium  carbonate, 
a  little  at  a  time,  stirring  thoroughly  and  heating,  and  con- 
tinue adding  the  carbonate  until  the  fluid  no  longer  has  an 
acid  reaction,  then  filter,  and  wash  once.  Decolorize  the 
filtrate,  which  is  usually  yellow-colored,  by  warming  with 
some  bone-black,  and  filter  again.  We  must  now  separate 
the  urea  from  the  barium  nitrate  formed.  This  is  done  by 
evaporating  to  dryness  on  the  water-bath  and  extracting 
the  residue  with  alcohol,  in  which  only  the  urea  is  soluble. 
The  alcoholic  solution  is  then  filtered  and  evaporated  to 
crj^stallizatiori.  The  crystals  of  urea  are  left  till  next  day, 
separated  from  the  mother-liquor,  pressed  dry  between  filter- 
paper,  and  purified  by  recrystallizing  from  a  small  quantity 
of  absolute  alcohol  (by  warming  in  a  flask). 

ATXT 

Urea,  OC  <  ATTT^  the  amide  of  carbonic  acid,  hence  also 

called  'carbamide,"  crystallizes  in  long  quadratic  prisms  or, 
when  crystalhzed  rapidly,  in  needles;  it  is  very  readily  sol- 
uble in  water  (at  100°  in  every  proportion),  less  readily, 
though  still  quite  soluble,  in  alcohol,  and  insoluble  in  anhy- 
drous ether.  It  is  not  precipitated  by  metallic  salts,  with  the 
exception  of  mercuric  nitrate. 

Reactions  of  Urea. 

1.  Heat  a  small  portion  in  a  dry  test-tube.  It  melts 
(melting-point  132°),  giving  a  strong  odor  of  ammonia,  and 
if  the  urea  is  not  dry  ammonium  carbonate  sublimes.  Con- 
tinue the  heating  till  the  melted  mass  just  begins  to  solidify 
(the  re-formation  of  the  solid  is  due  to  the  change  into  cyan- 
uric  acid).  When  heated  just  beyond  its  melting-point  urea 
forms  biuret: 


98     PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

0C<  /NH^ 

\NH2    OCX 
+         =       >NH+NH3 
/NH2    0C< 

\ATTT 


^NH. 


Biuret  gives  a  characteristic  reaction.  Dissolve  the 
fused  mass  in  water  with  the  addition  of  some  caustic  soda, 
then  add  cautiously  dilute  copper  sulphate  solution;  the 
copper  hydroxide  resulting  dissolves,  forming  a  reddish-violet 
fluid  (biuret  reaction). 

2.  Repeat  the  fusing  of  the  urea,  but  continue  the  heating 
until  the  entire  mass  has  again  solidified;  after  cooling  dis- 
solve in  water  containing  some  caustic  soda,  and  acidify 
cautiously  with  hydrochloric  acid:  precipitation  of  cyanuric 
acid,  C3O3N3H3  (cyanic  acid  is  first  formed;  this,  however, 
polymerizes  at  once  to  cyanuric  acid). 

3.  Dissolve  a  few  crystals  of  urea  on  a  watch-glass  in  a 
drop  or  two  of  water,  and  add  a  little  concentrated  oxalic  acid 
solution:  precipitation  of  urea  oxalate  (OCNaHJg -0211204 + 
H2O.     Examine  under  the  microscope. 

4.  Repeat  this  experiment,  using  nitric  acid  instead  of 
oxalic  acid:  urea  nitrate,  OCNgHi-HNOg,  (see  Fig.  7). 

5.  Heat  some  urea  with  caustic  soda  solution :  strong  evo- 
lution of  ammonia  with  the  formation  of  sodium  carbonate: 

OC<^^J+2NaOH  =  OC<^^^+2NH3. 

6.  Warm  a  small  drop  of  mercury  with  nitric  acid  in  a 
test-tube  and  then  add  a  little  urea:  marked  foaming  due 
to  the  development  of  a  colorless,  odorless  gas,  a  mixture  of 
carbon  dioxide  and  nitrogen.  The  reaction  is  due  to  the 
action  of  nitrous  acid  on  urea  • 

0CN2H,+  N2O3  =  CO2+ 2N2+ 2H2O. 


EXAMIXATIOX  OF   THE   URINE. 


99 


7.  Add  to  a  little  bromine-water  in  a  test-tube  an  ex- 
cess of  caustic  soda  solution.  Sodium  hypobromite,  NaBrO, 
is  formed.  If  some  m-ea  solution  is  added  to  this  mixture 
nitrogen  is  evolved,  causing  marked  effervescence,  while  the 


Fig.  7. — Urea  Nitrate. 

carbon  dioxide  formed  at  the  same  time  is  absorbed  by  the 
excess  of  caustic  soda  present: 

OCXoH,  +  SNaBrO  =  CO^  +  N^ + 2H,0  +  3XaBr. 

IV.   PREPARATION   OF   URIC   ACID,   C5H4N4O3. 

(a)  From  Urine.  Add  50  cc.  of  hydrochloric  acid  to  500  cc. 
of  urine  and  let  stand  for  twenty-four  hours  in  a  cool  place, 
filter,  and  wash  with  water.  Examine  the  crystals  of  uric 
acid  under  the  microscope.  Nearly  neutralize  the  filtrate 
with  annnonia,  then  add  magnesia  mixture,  filter,  and  add  to 
the  filtrate  some  silver  nitrate  solution:  precipitate  of  a 
double  compound  of  silver  and  magnesium  with  that  part  of 
the  uric  acid  which  was  not  precipitated  by  the  hydrochloric 
acid.  Filter  off  the  precipitate,  wash,  suspend  in  water, 
decompose  by  means  of  hydrogen  sulphide,  filter  from  the 
silver  sulphide,  evaporate  to  a  small  volume,  and  add  hydro- 


100  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

chloric  acid:   precipitate  of  uric  acid  (see  Quantitative  De- 
termination of  Uric  Acid,  page  188). 


Ftg.  8. — Uric  Acid :   (o)  from  alkali  urate  by  the  addition  of  hydro- 
icchlor  acid ;    (6)  spontaneously  deposited  from  urine. 

(6)  From  Guano  or  Snake  Excrement.  Heat  to  boiling 
50  g.  of  finely  powdered  guano  with  500  cc.  of  water  and  100  cc. 
of  caustic  soda  solution  (marked  foaming  and  evolution  of 
ammonia;  the  experiment  is  therefore  best  performed  under 
the  hood).  Keep  boiling  for  some  time,  replacing  the  water 
which  evaporates  by  hot  water,  until  the  greater  part  of  the 
guano  has  dissolved,  then  filter.  Pour  the  filtrate  into  about 
300  cc.  of  dilute  sulphuric  acid  (20  per  cent.)  contained  in  a 
porcelain  dish,  and  heat  to  the  boiling-point.  Continue  the 
heating  until  the  fluid  begins  to  bump  and  a  crystalline  sedi- 
ment precipitates.  Examine  under  the  microscope  to  see 
that  it  has  no  amorphous  sodium  urate  mixed  with  it.  If 
it  has,  then  more  acid  must  be  added  and  the  heating  con- 
tinued with  vigorous  stirring  or  on  the  water-bath.  Let  cool, 
filter,  wash  with  water  until  the  filtrate  no  longer  gives  a  pre- 
cipitate with  barium  chloride  or  only  a  faint  turbidity,  drain 
thoroughly,  and  dry  on  filter-paper. 


EXAMINATION  OF  THE   URINE.  101 

If  snake  urine,  so-called  excrement,  is  available,  10  g.  of 
this  and  20  cc.  of  caustic  soda  solution  are  sufficient. 

Uric  acid,  C3H4N4O3,  forms  a  crystalline  powder  extremely 
difficultly  soluble  in  water  (in  1800  parts  of  hot,  14,000  '  parts 
of  cold  water),  insoluble  in  alcohol. 

Reactions  of  Uric  Acid. 

1.  Place  a  small  quantity  of  uric  acid  with  a  little  water 
on  a  microscope-slide  and  treat  with  some  caustic  soda 
solution  or  piperazine  solution  (10  per  cent.);  the  crystals 
dissDlve.  When  all  or  nearly  all  has  dissolved  add  a  little 
hydrochloric  acid:  the  uric  acid  separates  in  characteristic 
spindle-shaped  crystals.     Examine  under  the  microscope. 

2.  Murexide  Test.  Pour  upon  a  very  small  quantity  of 
uric  acid  on  a  porcelain  crucible-cover  some  drops  of  nitric 
acid,  dissolve  by  warming  and  evaporate  cautiously,  avoid- 
ing heating  too  strongly:  there  remains  a  yellow  to  red  resi- 
due. Let  cool  and  moisten  the  residue  with  an  extremely 
small  quantity  of  ammonia:  purple-red  color  due  to  the 
formation  of  murexide.  Now  add  a  drop  of  caustic  soda 
solution:  deep-blue  color.  If  now  we  heat  again,  the  color 
becomes  paler  and  disappears  even  before  the  mass  becomes 
completely  dry.  If  the  purple-red  residue  or  the  blue  residue 
be  moistened  while  still  hot  with  a  few  drops  of  water,  it  dis- 
solves to  an  almost  colorless  fluid.  If  this  is  evaporated  by 
heating  with  a  very  small  flame  the  color  is  not  restored,  as 
the  murexide  is  very  easily  destroyed  (distinction  from  the 
reaction  of  the  xanthine  bases,  especially  of  guanine). 

3.  Dissolve  some  uric  acid  in  a  solution  of  sodium  car- 
bonate and  moisten  with  it  a  strip  of  paper  which  has  pre- 
viously been  saturated  with  a  solution  of  silver  nitrate:  spots 

*  According  to  His  and  Paul,  Zeitschr.  f.  physiol.  Chem.  31,  1  (1900- 
1901),  1  part  dissolves  in  39,500  parts  of  pure  water  at  18°.— O. 


102  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

of  reduced  silver  appear  at  once.  These  are  yellow-brown 
to  deep  black  according  to  the  quantity  of  the  dissolved 
uric  acid. 

4.  If  we  add  to  the  sodium  carbonate  solution  of  uric 
acid  some  magnesia  mixture  (solution  of  magnesium  hydrox- 
ide in  ammonia  and  ammonium  chloride)  and  then  add  silver 
nitrate  solution,  a  double  compound,  silver  magnesium  urate, 
separates  in  the  form  of  a  gelatinous  precipitate. 

5.  Dissolve  some  uric  acid  in  water  and  caustic  soda,  add 
some  Fehling's  solution  and  heat:  white  cuprous  urate  pre- 
cipitates, or,  when  the  quantity  of  the  copper  relative  to  the 
uric  acid  is  sufficiently  large,  red  cuprous  oxide  is  formed. 
This  reaction  is  important,  as  it  shows  that,  when  making 
the  Trommer's  test  with  normal  urine,  the  reducing  action 
of  the  uric  acid  always  present  must  be  taken  into  account. 

Of  the  reactions  the  precipitation  of  the  magnesium  silver 
urate  is  especially  important  for  the  isolation  of  uric  acid, 
the  murexide  test  for  its  detection.  Uric  acid  is  not  always 
deposited  from  urine  on  the  addition  of  hydrochloric  acid; 
it  is  then  necessary  to  use  the  precipitation  of  the  magnesium 
silver  c  mpound  to  show  its  presence. 

V.  DETECTION  OF  CREATININE,  C4H7N3O. 

Make  240  cc.  of  urine  faintly  alkaline  by  cautiously  adding 
milk  of  lime  and  precipitate  exactly  with  a  solution  of  cal- 
cium chloride;  dilute  with  water  to  300  cc,  mix  thoroughly, 
and  after  fifteen  minutes  filter  through  a  dry  filter.  Measure 
off  250  cc.  of  the  filtrate,  which  must  have  a  faintly  alkaline 
reaction,  and  after  neutralizing  with  acetic  acid  evaporate 
to  about  20  cc,  at  first  over  a  free  flame  and  then  on  the 
water-bath.  Mix  this  with  the  same  volume  of  absolute 
alcohol,  transfer  the  mixture  to  a  100-cc.  measuring-flask, 
rinsing  with  absolute  alcohol,  and  finally  fill  the  flask  up  to 


EXAMINATION  OF  THE  URINE. 


103 


the  mark  with  tlie  same  Hquid.  Let  stand  till  the  next  day, 
filter  through  a  dry  filter,  and  add  to  the  filtrate  about  twenty 
drops  of  an  alcoholic  solution  of  zinc  chloride.  After  stand- 
ing for  one  or  two  days  crystals  of  creatinine  zinc  chloride, 
(C4H-XjO)2ZnCl2,  ^^ill  be  deposited.  Examine  these  under 
the  microscope.     Filter,  and  wash  with  alcohol. 

For  identification  Weyl's  reaction  may  be  ased:  Grind 
the  creatinine  zinc  chloride  to  a  fine  powder,  boil  a  small 
quantity  of  this  with  water  in  a  test-tube,  let  cool  and  filter. 
Add  to  the  filtrate  a  few  drops  of  a  very  dilute  solution  of 
sodium  nitroprusside  (freshly  made)  and  then  some  caustic 
soda  solution:  deep-red  color,  which  quickly  fades  to  a 
straw-yellow. 


Fig.  9. — Creatinine  ZLac  Chloride  from  Urine. 

The  Weyl  reaction  may  also  be  used  directly  with  the 
urine.  As  the  urine  often  contains  acetone  in  considerable 
quantity  and  this  gives  a  very  similar  test,  it  is  advisable 
before  trying  the  reaction  to  boil  the  urine  for  a  few  minutes, 
thus  driving  off  the  acetone  and  then  cool. 

Jaffe's  reaction  with  picric  acid  may  also  be  used  directly 
with  the  urine:  Add  to  the  urine  some  aqueous  picric  acid 
solution  and  then  a  few  drops  of  caustic  soda:  deep-red  color. 


104  PHYSIOLOGICAL  AND   PATHOLOGICAL  CHEMISTRY. 

VI.  Detection  of  Oxalic  Acid. 

Evaporate  500  cc.  of  unfiltered  urine  over  a  small  free 
flame  to  about  150  cc.  After  cooling  add  20  cc.  of  hydro- 
chloric acid,  transfer  to  a  separating-funnel  and  shake  with 
about  an  equal  volume  of  a  mixture  of  alcohol  and  ether  (9 
volumes  of  ether,  1  volume  of  absolute  alcohol),  carefully 
separate  the  ether  extract  and  filter  it  through  a  dry  filter. 
Repeat  the  extraction  with  ether  once  more  and  distil  the 
combined  ether  extracts  from  a  dry  flask.  The  fluid  remain- 
ing in  the  flask  is  poured  into  a  dish,  the  flask  being  rinsed 
out  once  with  a  little  alcohol,  then  with  water,  and  the  mix- 
ture is  heated  on  the  water-bath  with  the  addition  of  some 
water  until  the  odor  of  alcohol  and  ether  has  disappeared. 
The  aqueous  fluid  remaining,  whose  volume  should  be  about 
20  cc,  is  cooled,  and  filtered  from  some  resinous  material 
which  separates.  Make  the  solution  faintly  alkahne  with 
ammonia,  add  1  or  2  cc.  of  a  10  per  cent,  calciimi  chloride 
solution,  and  acidify  with  acetic  acid.  The  white  precipitate 
of  calcium  oxalate,  which  forms  either  at  once  or  else  gradu- 
ally, is  amorphous,  but  quite  homogeneous  when  it  separates 
quickly.  It  is  crystalUne  when  precipitated  more  slowly, 
and  then  frequently  shows,  instead  of  octahedral  forms,  those 
described  by  Feser  and  Friedberger  ^  (quadratic  prisms  with 
pyramidal  end  faces). 

VII.  HiPPURic  Acid,  Benzoyl  Glycocoll, 

CH-NH(COCeH,) 

COOH 

To  about  300  cc.  of  horse-urine  add  milk  of  lime  till  the 
reaction  is  strongly  alkaline  (to  separate  phosphoric  acid 
and  a  part  of  the  coloring  matter),  filter,  evaporate  to  sirupy 

^  Maly's  Jahresber.  f.  Thierchemie,  4,  231. 


EXAMINATION  OF  THE  URINE. 


105 


consistency,  precipitate  with  alcohol,  filter,  evaporate  the 
alcoholic  extract  and,  when  perfectly  cold,  acidify  strongly 
with  hydrochloric  acid.  The  hippuric  acid  separates  as  a 
crystalline  paste.  Filter  this  off  (keeping  the  filtrate), 
wash,  drain  by  pressing  between  drying-paper,  dissolve  in 
water  to  which  ammonia  has  been  added,  decolorize  by  boil- 
ing with  some  bone-black,  filter,  concentrate,  precipitate 
again  by  adding  hydrochloric  acid,  filter,  wash  and  dry  on 
drying-paper  in  the  air.  Examine  a  small  portion  while  still 
moist  under  the  microscope.  If  it  proves  to  be  contaminated 
with  benzoic  acid  (irregularly  indented  leaflets,  see  Fig.  10), 


Fig.  10. — a,  benzoic  acid ;  b,  hippuric  acid. 

treat  the  dry  mixture  of  acids  with  ether  to  dissolve  the  ben- 
zoic acid.  In  order  to  obtain  larger  crystals  the  acid  may 
be  recrystallized  from  hot  water. 


Reactions  of  Hippuric  Acid. 

1,  A  small  portion  is  heated  in  a  test-tube  with  water:  it 
dissolves.  On  cooling  the  hippuric  acid  separates  in  needles. 
Examine  under  the  microscope. 

2,  Heat  a  small  portion  in  a  test-tube:   the  hippuric  acid 


106  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

melts,  at  first  without  decomposition  (melting-point  187°), 
When  heated  more  strongly  the  melted  mass  turns  red,  gives 
a  sublimate  of  benzoic  acid,  and  develops  an  odor  resembling 
that  of  the  oil  of  bitter  almonds  (benzonitrile,  CgHsCN,  and 
prussic  acid,  HCN). 

The  red  color  is  due  to  the  decomposition  of  the  glycocoll. 
After  the  tube  is  cold,  cut  it  off  close  below  the  subUmate  and 
place  the  upper  part  of  the  tube  in  a  weak  solution  of  sodium 
carbonate  in  a  test-tube.  To  the  solution  resulting  add  a 
Uttle  hydrochloric  acid:  precipitation  of  benzoic  acid. 
Examine  under  the  microscope. 

3.  Evaporate  a  small  quantity  of  the  crystals  of  hippuric 
acid  with  some  drops  of  firaiing  nitric  acid,  mix  the  residue 
with  some  sand,  put  it  into  a  tube  and  heat  strongly:  odor 
of  the  oil  of  bitter  almonds  due  to  the  formation  of  nitroben- 
zene, CeHjNOj  (Liicke's  reaction).  Benzoic  acid  and  many 
other  acids  of  the  aromatic  series  also  give  this  reaction. 

VIII.  PHENOL,  CeHsOH,  Cresol,  C^H^OH. 

PHENYL  SULPHURIC    ACID,    CRESYL   SULPHURIC    ACID. 

(a)  Phenol. 
Radiating  crystalline  mass  (if  previously  melted)  or  loose 
crystals,  forming  an  oily  fluid  with  one-tenth  its  volume  of 
water.     Melting-point  42° ;  readily  soluble  in  ether,  in  alco- 
hol, and  in  fifteen  parts  of  water. 

Reactions  of  Phenol. 

Use  a  2  per  cent,  and  a  0.2  per  cent,  solution  of  phenol 
and  make  parallel  tests.  The  conduct  given  in  the  following 
experiments  is  for  the  2  per  cent,  solution: 

1.  Addition  of  ferric  chloride  solution:  deep  blue  (ame- 
thyst-blue) color.  Strong  acids  discharge  the  color.  Many 
phenol  derivatives,  e.g.,  salicyhc  acid,  give  a  similar  reaction 
with  ferric  chloride. 


EXAMINATION  OF   THE   URINE.  107 

2.  Add  to  the  phenol  solution  one-fourth  its  volume  of 
ammonia,  then  a  few  drops  of  a  solution  of  chloride  of  Ume 
(bleaching-powder),  and  warm  gently,  but  not  to  boiUng:  blue 
or  green  color. 

3.^  Add  a  few  drops  of  Millon's  reagent  and  heat  to  boil- 
ing :  intense  dark-red  color  or  dark-red  precipitate.  The  reac- 
tion is  very  distinct  even  at  1  :  60,000,  but  at  such  great 
dilution  only  a  dehcate  rose-color  appears.  All  benzene 
derivatives  which  contain  a  hydroxyl  group  in  the  aromatic 
residue  give  a  similar  reaction  (0.  Nasse). 

4.  Addition  of  bromine-water  produces  at  first  a  gelatinous 
precipitate  of  monobromphenol  or  dibromphenol,  which  is 
characterized  by  a  very  penetrating  odor.  On  further  addi- 
tion of  bromine  yellowish-white  tribromphenol,  CeHjBrjOH, 
is  formed.  In  dilute  solutions  this  last  compound  is  at  once 
formed. 

Of  the  reactions,  3  and  4  are  the  most  dehcate  and  the 
most  used. 

After  the  medicinal  use  of  phenol  it  may  be  found  in  the 
urine.  Horse-urine  and  pathological  urine  contain  cresol 
instead  of  phenol,  or  at  least  in  much  larger  quantity.  The 
reactions  of  cresol  (paracresol)  are  similar  to  those  of  phenol, 
but  less  marked.  The  color  with  ferric  chloride  is  not  blue,' 
but  a  dirty  gray.  This  is  partly  due  to  the  fact  that  the 
cresol  is  far  less  soluble  in  water  than  the  phenol. 

(6)  Phenyl  Sulphuric  Acid  or  Cresyl  Sulphuric  Acid. 
Detection  of  Phenol  in  Urine. 
Phenol  and  cresol  are  never  found  as  such  in  urine,  but 
always  combined  with  sulphuric  acid  in  the  form  of  the  potas- 
sium salts  (so-called  conjugate  or  ethereal  sulphates), 

^  \0K        "^    ^^\0K 

Potassium  phenyl  sulphate        Potassium  cresyl  sulphate 


108  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

and  also  in  combination  with  glycuronic  acid.  To  isolate  the 
phenols  or  to  detect  their  presence,  these  sulphuric  acid  esters 
must  be  decomposed.  This  is  done  by  heating  with  hydro- 
chloric acid. 

1.  Detection  in  Horse-urine.  The  filtrate  obtained  in  the 
preparation  of  hippuric  acid  (see  VII,  page  105)  is  diluted  to 
200  cc,  and  150  cc.  distilled  off.  The  distillate  has  the  char- 
acteristic odor  of  paracresol.  It  frequently  contains  some 
benzoic  acid  in  crystalhne  form. 

A  part  of  the  cresol  may  be  used  for  the  reactions,  the  rest 
is  made  faintly  alkaline  with  sodium  carbonate  solution  and 
shaken  in  a  separating-funnel  with  a  little  ether,  the  ether 
separated  and  evaporated:  there  remains  a  mixture  of  cresol 
and  a  little  phenol. 

2.  Detection  in  Human  Urine.  Two  hundred  cubic  centi- 
meters of  urine  to  which  50  cc.  of  hydrochloric  acid  have  been 
added  are  distilled  until  a  small  portion  of  the  distillate  no 
longer  gives  any  turbidity  with  bromine-water.  If  we  wish 
to  detect  very  small  quantities  (in  normal  urine),  500  cc.  of 
the  urine  are  first  made  alkahne  with  sodium  carbonate  solu- 
tion and  evaporated  nearly  to  dryness,  the  residue  treated 
with  one-fifth  of  its  volmne  of  hydrochloric  acid  and  distilled 
(J.  Munk). 

Instead  of  the  roundabout  method  of  distillation  we  may 
make  use  of  a  shorter  procedure,  which  depends  on  the 
hydrolysis  by  nitric  acid  and  conversion  into  nitro  com- 
pounds. This  method  is  used  especially  to  decide  the  ques- 
tion whether  much  of  the  carbolic  acid  used  in  the  dressing 
of  wounds,  etc.,  is  absorbed.  In  this  case  a  parallel  experi- 
ment with  normal  urine  should  always  be  made. 

Add  to  the  urine  in  a  test-tube  some  nitric  acid  and  heat 
to  boiling :  an  odor  resembling  that  of  bitter  almonds  becomes 
perceptible  (formation  of  volatile  ortho-nitrophenol) ;  when 
perfectly  cold  add  bromine-water  to  part  of  the  liquid:  more 


EXAMINATION  OF    THE  URINE.  109 

or  less  marked  turbidity  or  precipitate  of  nitrotribromphenol. 
Normal  mine,  when  treated  in  the  same  way,  either  remains 
clear  or  gives  a  faint  turbidity.  Make  the  rest  of  the  hquid, 
obtained  after  heating  with  nitric  acid,  alkaline  with  caustic 
soda  solution:  orange-red  color  due  to  the  formation  of 
sodimn  nitrophenolate. 

IX.  Pyrocatechin,  C6H4(OH)2.(o.) 

Use  an  aqueous  solution  (0.1  g.  :  25-50  cc.)  of  the  com- 
mercial product  for  the  reactions. 

1.  Add  cautiously  some  dilute  ferric  chloride  solution: 
green  color.  If  now  a  trace  of  ammonia  be  added  or,  better, 
a  trace  of  tartaric  acid  and  then  anmionia,  the  green  color 
changes  to  violet.  Acidifying  with  acetic  acid  restores  the 
green  color, 

2.  Add  to  some  of  the  solution  a  Uttle  ammonia  and  then 
a  few  drops  of  silver  nitrate  solution:  almost  immediate  re- 
duction to  metallic  silver  results. 

3.  On  the  addition  of  caustic  soda  solution  the  hquid  takes 
up  oxygen  and  becomes  colored  green,  then  brown  and  black, 
especially  on  shaking. 

4.  Pyrocatechin  is  completely  precipitated  by  lead  acetate 
solution,  as  the  filtrate  gives  none  of  the  above  reactions. 

X.  Indigo  Blue,    Indican  (Potassium  Indoxyl 

SULPHATE),    03S<'q^«^«^. 

(a)  Indigo  Blue,  C16H10N2O2. 

1.  Heat  cautiously  some  finely  powdered  commercial 
indigo,  spread  out  in  a  porcelain  or  metal  dish.  The  upper 
surface  becomes  covered  with  purple  crystals  of  indigo  blue. 

2.  Heat  a  small  portion  in  a  dry  test-tube.  The  tube 
becomes  filled  with  a  purple  vapor,  resembling  the  vapor  of 


110  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

iodine.  A  part  of  the  indigo  carbonizes,  another  part  sub- 
limes; in  this  process,  however,  some  of  the  indigo  blue  is 
always  transformed  into  the  isomeric  indigo  red  (Rosin), 

3.  Heat  a  little  indigo  with  some  chloroform:  blue  solu- 
tion. 

4.  Heat  a  little  indigo  with  concentrated  sulphuric  acid, 
let  cool  completely,  and  then  pour  into  water:  blue  solution 
of  indigo  mono-  and  di-sulphonic  acids.  Filter  the  solution 
and  examine  with  the  spectroscope:  strong  absorption-band 
between  C  and  D,  nearer  to  D. 

(6)  Detection  of  Indican. 

1.  Jaffa's  Indican  Test.  Add  to  some  of  the  urine  an  equal 
volume  of  hydrochloric  acid,  then,  drop  by  drop,  with  con- 
stant shaking,  a  dilute  solution  of  chloride  of  lime  (bleaching- 
powder)  (1:20).  Then  add  about  1  cc.  of  chloroform  and 
shake  gently :  the  chloroform  becomes  colored  blue,  owing  to 
its  dissolving  the  indigo  formed.  The  quantity  of  the  chlo- 
ride of  lime  is  difficult  to  estimate,  and  an  excess  may  oxidize 
the  indigo  blue.  The  reaction  always  take  place  slowly  and 
requires  some  time.  Urine  which  is  rich  in  indican  turns 
green  or  even  blue  directly,  while  urine  poor  in  indican  does 
not  do  this.  Frequently,  instead  of  a  blue,  a  violet  color 
results,  which  is  not  taken  up  by  the  chloroform.  This  is  due, 
according  to  Rosin,  to  indigo  red  or  to  urorosein.  Normal 
urine  as  a  rule  turns  violet  to  red-violet,  but  it  gives  up  indigo 
blue  to  the  chloroform.  Strongly  colored  urines,  e.g.,  icteric, 
must  be  decolorized  by  the  addition  of  a  little  basic  lead  ace- 
tate and  filtered  before  making  the  test. 

2.  Modification  of  the  Indican  Test  according  to  Obermayer. 
Precipitate  the  urine  with  the  basic  lead  acetate  solution,  tak- 
ing care  not  to  use  an  excess,  filter  through  a  dry  filter,  shake 
the  filtrate  vigorously  for  one  to  two  minutes  with  an  equal 
volume   of  fuming  hydrochloric   acid    (containing  in    1000 


EXAMINATION  OF  THE   URINE.  HI 

parts  two  to  four  parts  of  ferric  chloride),  and  then  mth  1  cc. 
of  chloroform.  The  ferric  chloride  is  preferable  to  the  chlo- 
ride of  lime,  as  a  loss  of  indigo  by  oxidation  cannot  then  take 
place. 

If  the  urine  contains  alkaline  iodides,  then,  after  making 
the  test,  add  a  few  drops  of  a  10  per  cent,  solution  of  sodium 
thiosulphate  to  combine  with  the  iodine. 

XI.     UROBILIN. 

Discovered  by  Jaffe  in  urine  (febrile  urine,  retained  urine). 
Detection: 

1.  Some  of  the  urine  is  examined  with  the  spectroscope: 
urines  which  contain  considerable  urobilin  often  show  directly 
the  characteristic  absorption-band  at  the  border  of  the  green 
and  blue,  between  the  lines  b  and  F  (see  Table  of  Absorption 
Spectra,  No.  7).  Sometimes  the  absorption-band  is  more 
distinct  after  the  addition  of  a  few  drops  of  hydrochloric  acid. 
Not  infrequently  the  absorption  of  light  is  so  strong  that  the 
urine  must  be  diluted  with  several  times  its  volume  of  water 
in  order  to  obtain  a  distinct  absorption-band, 

2.  Add  ammonia  to  a  second  portion  of  the  urine,  filter 
from  the  precipitated  phosphates  after  a  few  minutes,  and 
add  some  drops  of  zinc  chloride  solution  to  the  filtrate:  with 
urine  containing  considerable  urobilin  a  green  fluorescence 
becomes  perceptible.  The  spectroscopic  examination  shows 
almost  the  same  absorption-band  (somewhat  nearer  to  b). 

3.  Ten  to  twenty  cubic  centimeters  of  urine  are  acidified 
with  a  few  drops  of  hydrochloric  acid  and  then  shaken  gently 
with  5  to  10  cc.  of  amyl  alcohol.  Examine  the  amyl  alcohol 
extract  with  the  spectroscope.  On  the  addition  of  a  few 
drops  of  zinc  chloride  solution  (1  g.  zinc  chloride  in  100  cc.  of 
ammoniacal  alcohol)  the  extract  shows  fluorescence  (Nencki 
and  Rotschy). 

4.  To  a  fourth  portion  of  the  urine  add  a  few  cubic  centi- 


112  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

meters  of  chloroform,  mix  repeatedly,  avoiding  shaking  too 
vigorously,  separate  the  chloroform,  and  add  to  it  a  drop  of 
an  alcoholic  solution  of  zinc  chloride;  any  turbidity  is  to  be 
cleared  up  by  the  addition  of  absolute  alcohol:  the  chloro- 
form becomes  rose-red  with  a  greenish  fluorescence  (E,  Wir- 
sing  0- 

5.  Urine  containing  a  considerable  quantity  of  urobilin 
gives  the  biuret  reaction  with  caustic  soda  and  copper  sul- 
phate solutions  (confusion  with  albumoses  and  peptones). 

If  the  urobilin  cannot  be  detected  by  the  above  methods 
proceed  as  follows: 

1.  Precipitate  200  cc.  of  urine  completely  with  basic  lead 
acetate,  filter,  wash  once  wdth  water,  dry  the  precipitate  at 
room  temperature,  grind  it  in  a  mortar  with  alcohol  and  5  g. 
of  oxalic  acid,  let  stand  twelve  to  twenty-four  hours,  and 
filter  (if  absolute  alcohol  is  used,  the  drying  of  the  precipi- 
tate may  be  dispensed  with;  it  is  then  sufficient  to  drain  it 
thoroughly  on  filter-paper).  Make  a  portion  of  the  filtrate 
alkaline  with  ammonia,  filter  from  the  ammonium  oxalate 
which  separates,  and  add  a  drop  of  zinc  chloride  solution: 
green  fluorescence,  absorption-band.  Not  infrequently,  how- 
ever, these  reactions  are  indistinct.  In  this  case,  in  order  to 
purify  the  rest  of  the  alcoholic  filtrate,  shake  it  in  a  sepa- 
rating-funnel  with  about  20  cc.  of  chloroform  and  enough 
water  so  that  the  chloroform  settles  readily.  Separate  the 
chloroform,  filter  it  through  a  dry  filter,  and  examine  spec- 
troscopically  both  before  and  after  the  addition  of  a  drop  of 
an  alcoholic  solution  of  zinc  chloride. 

The  urine  must  not  be  allowed  to  stand  too  long  before 
it  is  examined,  as  the  urobilin  changes  on  standing  into  a 
modification  which  lacks  the  most  essential  properties  of 
this  pigment.  This  takes  place  even  with  urine  containing 
a  considerable  quantity  of  urobilin. 

1  Verhandl.  d.  Wiirzb.  phys.-med.  Gesellsch.  N.  F.  26,  No.  3. 


EXAMINATION  OF  THE  URINE.  113 

2.  Fifty  cubic  centimeters  of  urine  are  gently  shaken  with 
the  same  quantity  of  perfectly  pure  ether,  which  must 
contain  no  alcohol  or  acid;  the  ethereal  extract  is  then 
evaporated  and  the  residue  dissolved  in  2  to  3  cc.  of 
absolute  alcohol.  The  solution  shows  the  green  fluorescence 
and  the  absorption-band.  Its  color,  curiously  enough,  is 
often  pure  yellow. 

XII.  Detection  of  unoxidized  Sulphur. 

Pour  some  hydrochloric  acid  on  a  small  piece  of  zinc  in  a 
dish,  let  the  acid  act  for  a  short  time,  then  pour  off  the  hydro- 
chloric acid  and  rinse  the  zinc  with  water.  Put  the  zinc  in 
a  flask  with  about  50  cc.  of  urine,  add  enough  hydrochloric 
acid  to  cause  an  evolution  of  hydrogen,  and  fasten  a  strip  of 
filter-paper,  which  has  been  previously  moistened  with  lead 
acetate  solution,  in  the  neck  of  the  flask  by  means  of  a  loosely 
fitting  cork.  The  paper  turns  browTi  or  black  after  some 
time  from  the  formation  of  lead  sulphide.  Only  the  neutral 
sulphur  of  the  urine  forms  hydrogen  sulphide  with  nascent 
hydrogen.  Neither  the  sulphuric  acid  nor  the  ethereal  sul- 
phates are  acted  on. 

XIII.  Detection  of  Pepsin. 

Divide  the  urine  to  be  examined,  about  50  cc,  into  two 
equal  parts,  heat  one-half  to  boiling,  and  let  cool;  the  second 
half  is  not  heated  To  each  of  the  two  portions  add  4  or  5 
drops  of  hydrochloric  acid.  Ten  cubic  centimeters  of  the 
urine  thus  prepared  are  placed  in  a  test-tube  with  a  shred  of 
fibrin  and  digested  at  40°.  It  is  advisable  to  make  two  tests 
with  each  portion  of  the  urine.  The  shred  of  fibrin  dissolves 
in  the  unboiled  portion  of  the  urine  in  a  longer  or  shorter 
time  according  to  the  amount  of  pepsin  present,  but  not  in 
the  boiled  portion.     The  unboiled  portion  of  the  urine  in 


114  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

which  the  fibrin  dissolved  gives  the  biuret  reaction  (after  it 
has  been  neutrahzed  with  dilute  sodium  carbonate  solution, 
boiled  and  filtered)  while  the  boiled  portion  does  not. 

XrV.     DETECTION  OF  ALBUMIN. 

1.  Heat  a  portion  of  the  clear,  filtered  urine  to  boiling.  If 
it  remains  clear  and  the  reaction  is  acid,  there  is  no  albumin 
present.  If  it  becomes  cloudy,  the  turbidity  may  be  due  to 
the  precipitation  either  of  albumin  or  of  calcium  phosphate. 
To  decide  this,  acidify  quite  faintly  with  acetic  acid :  if  the 
urine  then  becomes  clear,  the  turbidity  is  due  to  calcium  phos- 
phate and  the  urine  is  free  from  albumin;  if,  on  the  other 
tand,  the  turbidity  remains  and  the  precipitate  tends  to  col- 
lect in  flakes  on  standing,  the  urine  contains  albumin.  A 
doubt  can  only  arise  when  the  turbidity  which  remains  is  very 
small  and  uniform.  In  this  case  it  may  be  due  to  the  pres- 
fence  of  mucin  or  nucleoalbumin  in  the  urine.  This  is  to  be 
assumed  if  the  urine  becomes  cloudy  even  in  the  cold,  when 
acidified  directly  with  acetic  acid  or  after  dilution  with  an 
equal  volume  of  water.  If  the  urine  remains  clear  on  boil- 
ing, but  reacts  strongly  alkaline  (a  rare  case),  it  may  contain 
albumin.  In  this  case  also  the  addition  of  acetic  acid  to  acid 
reaction  decides  the  matter. 

2.  Instead  of  acetic  acid  we  may  use  nitric  acid  to  acidify 
the  boiled  urine. 

3.  Add  to  the  urine  one-third  of  its  volume  of  nitric  acid. 
If  it  remains  clear  (when  only  traces  of  albumin  are  present 
the  turbidity  appears  very  gradually),  then  it  is  free  from 
albumin;  if  it  becomes  cloudy,  the  cloudiness  may  be  due  to 
albumin  or  to  urates,  albumoses,  or  resin  acids  (after  the  use 
of  balsams  or  sandal-wood  oil).  If  the  cloudiness  remains  on 
warming,  then  it  is  due  to  albumin. 

4.  Add  one-third  the  volume  of  concentrated  sodium 
chloride  solution,  acidify  with  acetic  acid  to  distinctly  acid 


EXAMINATION  OF  THE   URINE.  115 

reaction,  and  heat  to  boiling.  A  turbidity  or  precipitate 
shows  the  presence  of  albumin  (mucin  remains  in  solution). 

5.  Acidify  with  acetic  acid  and  add  a  few  drops  of  potas- 
sium ferrocyanide  solution:  cloudiness  proves  the  presence 
of  albumin  (a  very  delicate  reaction,  but  only  available  when 
the  urine  remains  clear  after  the  addition  of  acetic  acid  alone; 
moreover,  it  is  also  given  by  albumose). 

The  precipitated  albumin  may  be  filtered  off,  washed,  and 
used  for  the  color  reactions  (see  page  4). 

The  examination  for  both  globulin  and  albumin  is  carried 
out  as  in  the  case  of  blood-serum  (page  59),  the  urine  being 
previously  made  alkaline  with  ammonia  and  filtered. 

XV.    DETECTION  OF  ALBUMOSES  (PEPTONE). 

To  detect  albumoses  (peptone)  we  precipitate  with  phos- 
photungstic  acid  according  to  Hofmeister  and  try  the  biuret 
reaction  with  the  precipitate,  after  the  albumoses  have  been 
set  free  by  means  of  baryta  or  (simpler)  by  caustic  soda.  The 
presence  of  urobilin,  which  is  carried  down  in  the  precipitate 
'W'ith  phosphotungstic  acid  and  also  gives  the  biuret  reaction, 
complicates  matters.  If  any  considerable  amount  of  uro- 
bilin is  present,  it  must  be  removed  (most  readily  with  abso- 
lute alcohol)  before  trying  the  biuret  reaction  with  the  pre- 
cipitate. When  the  amount  of  urobilin  is  small  and  the 
urine  is  not  highly  colored,  this  may  be  dispensed  with  and 
method  1  may  be  used. 

1.  Acidify  50  cc.  of  urine  (for  practice  use  urine  to  which 
0.25  to  0.5  g.  of  the  commercial  peptone  to  the  liter  has  been 
added)  in  a  beaker  with  a  few  cubic  centimeters  of  hydro- 
chloric acid,  precipitate  with  phosphotungstic  acid,  and  heat 
on  the  wire  gauze.  In  a  few  minutes  the  precipitate  forms  a 
resinous  mass,  which  sticks  to  the  bottom  of  the  vessel.  As 
soon  as  this  has  occurred,  pour  off  the  supernatant,  nearly 
clear,  liquid  as  completely  as  possible  and  rinse  the  resinous 


116  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

mass,  which  later  becomes  brittle,  twice  with  distilled  water. 
With  some  care  this  may  easily  be  done  almost  without  any 
loss.  Pom-  over  the  precipitate  a  few  cubic  centimeters  of 
water  and  dissolve  it  by  adding  sodium  hydroxide  solution. 
The  deep-blue  solution  is  warmed  on  the  wire  gauze  until  the 
color  has  disappeared,  then  poured  into  a  test-tube,  cooled, 
and  copper  sulphate  solution  cautiously  added.  The  color  is 
not  always  a  pure  violet,  but  often  only  a  dirty  red  or  even 
yellowish  red.  Often  10  to  15  cc.  of  the  urine  will  give  the 
reaction. 

If  the  urine  contains  albumin  it  must  previously  be  treated 
with  sodium  acetate  and  enough  ferric  chloride  to  give  the 
fluid  a  blood-red  color,  the  acid  neutralized  with  dilute 
sodium  hydroxide  solution  and  heated  to  boiling.  The 
filtrate  must  give  neither  a  turbidity  nor  a  blue  color  with 
acetic  acid  and  potassium  ferrocyanide  (a  very  faint  blue 
color  is  very  difficult  to  avoid  and  may  be  disregarded). 

If  the  urine  contains  mucin  or  nucleoalbumin  it  must  be 
precipitated  with  a  little  neutral  lead  acetate  solution,  so 
that  a  heavy  flocculent  precipitate  is  formed.  Then  after 
filtration  precipitate  with  phosphotungstic  acid,  either  at 
once  or  after  treatment  with  sodium  acetate  and  ferric  chlo- 
ride in  case  the  urine  also  contains  albumin.  In  the  case  of 
animal  urines  the  previous  treatment  with  lead  acetate  solu- 
tion is  advisable. 

2.  For  urines  containing  considerable  urobilin  v.  Aldor  ^ 
has  modified  this  method  as  follows:  8  to  10  cc.  of  urine  are 
made  acid  with  a  few  drops  of  hydrochloric  acid,  precipitated 
with  phosphotungstic  acid  and  centrifuged.  The  super- 
natant fluid  is  poured  off,  the  precipitate  covered  with  abso- 
lute alcohol  and  again  centrifuged.  This  is  repeated  until 
the  alcohol  remains  entirely  colorless,  then  suspend  the  pre- 

*  Berliner  Klinische  Wochenschr.,  1899,  Nos.  35  and  36. 


EXAMINATION  OF  THE  URINE.  H7 

cipitate  in  water  and  dissolve  by  adding  sodium  hydroxide 
solution,  etc.  If  a  centrifuge  is  not  available  the  precipitate 
may  be  washed  wth  alcohol  on  the  filter. 

3.  A*  larger  amount  of  primary  albumoses  CO. 3  g.  com- 
mercial peptone  to  100  cc.  of  urine)  may  be  detectf^d  as  fol- 
lows: Acidify  the  urine  with  acetic  acid  to  distinctly  acid 
reaction  and  add  an  equal  volume  of  concentrated  salt  solu- 
tion: turbidity,  which  clears  up  on  heating  and  returns  on 
cooling. 

XVI.  Glucose,  CB.,/^^ 

Also  called  grape-sugar,  diabetic  sugar,  and  dextrose, 
easily  soluble  in  water  and  dilute  alcohol,  difficultly  in  absf>- 
lute  alcohol.  Melting-point  of  the  anhydrous  glucos^i  *  146°. 
The  solution  of  glucose  is  dextrorotator}'. 

1.  A  small  portion  is  cautiously  heated  in  a  dry  test-tube. 
The  glucose  melts  and  turns  yellow;  when  heated  more 
strongly  the  melted  mass  turns  dark  browTi  and  develops  a 
peculiar  odor — so-called  caramel  odor.  After  cooling  dis- 
solve the  residue  in  water:  deep-brown  solution  (caramel 
color  ased  in  confectioner}'). 

2.  Place  a  small  quantity  of  glucose  and  a  piece  of  caustic 
potash  in  a  test-tube,  add  a  few  drops  of  water,  and  heat: 
\'igorous  reaction  and  a  browTi  coloration.  When  perfectly 
cold  acidify  cautiously  with  dilute  sulphuric  acid:  odor  of 
caramel. 

3.  Trommer's  test;  4.  Bismuth  test;  5.  Potassium  fer- 
rocvanide  test;  6.  Silver  test;  7.  Indigo  test;  8.  Rubner's 
test;  and  9.  Fermentation  test.  See  under  Milk-sugar, 
page  11.  For  all  these  reactions  use  two  solutions  A  and  B, 
A  containing  4  g.  of  glucose  dissolved  in  200  cc.  of  water,  i.e., 

>  Glucose  also  crystallizea  with  one  molecule  of  water  of  crystallusa- 
tion:    CH„0.-|- H,0, 


118  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

2  per  cent.;  and  B  20  cc.  of  A  diluted  to  200  cc,  i.e.,  0.2  per 
cent. 

Experiment  7  is  to  be  made  only  with  solution  B. 

10.  Phenyl  Hydrazine  Test. — Dissolve  by  shaking  2.5  g. 
of  phenyl  hydrazine  hydrochloride  and  5  g.  of  sodium  acetate 
in  100  cc.  of  a  1  per  cent,  glucose  solution  or  add  about  2  cc. 
of  phenyl  hydrazine,  which  has  been  previously  dissolved  in 
acetic  acid  to  acid  reaction,  filter  if  necessary,  heat  for  three- 
quarters  of  an  hour  upon  the  water-bath,  and  let  cool  gradu- 
ally: crystalline  mass  composed  of  bright  yellow  needles  of 
phenyl  glucosazone,  C18H22N4O4.  Filter  these  off,  wash,  dry  a 
small  portion,  and  determine  the  melting-point.  This  should 
be  204°  to  205°,  if  the  compound  is  quite  pure.  If  it  is  found 
to  be  considerably  lower,  as  is  frequently  the  case  with  dia- 
betic urine,  or  too  high,  which  happens  also  sometimes,  the 
compound  should  be  recrystallized  from  a  mixture  of  equal 
volumes  of  water  and  alcohol.  Most  of  the  sugars  yield 
osazones.  These  are  usually  characterized  by  their  melting- 
points. 

11.  Reaction  of  Molisch. — To  ten  drops  or  0.5  cc.  of  the 
sugar  solutions  (0.2  per  cent,  and  0.02  per  cent.)  add  one 
drop  of  an  alcoholic  or  methyl  alcoholic  solution  of  a-naph- 
thol,  and  let  1  cc.  of  pure  concentrated  sulphuric  acid  cau- 
tiously run  down  the  wall  of  the  test-tube.  At  the  surface 
of  contact  of  the  two  fluids  a  beautiful  violet-red  ring  will 
form,  which,  on  shaking  gently,  increases  in  breadth  and 
intensity.  The  reaction  of  Molisch  is  a  general  one  and  is 
common  not  only  to  the  sugars,  but  to  all  carbohydrates. 

Detection  of  Glucose  in  Urine. 

Dissolve  4  g.  of  glucose  in  100  cc.  of  a  urine  of  moderate 
concentration  (specific  gravity  not  less  than  1017) :  solution 
A.  Mix  10  cc.  of  solution  A  with  90  cc,  of  the  same  urine: 
solution  B.    A  part  of  the  urine  is  kept  for  check  experi- 


EXAMINATIOX  OF  THE    URINE.       .  119 

merits.  Trj^  the  Trommer's  test,  the  bismuth  test,  the  silver 
test,  and  the  indigo  test  with  these  solutions  and  ^vith  the 
urine  itself  as  a  check.  Tronuner's  test  is  to  be  made  both 
with  a  large  and  with  a  small  quantity  of  copper  sulphate 
solution. 

The  following  experiments  are  to  be  made  only  with  the 
weaker  solution  B  and  with  the  urine  as  a  check : 

1.  Fermentation  Test. 

2.  Phenyl  Hydrazine  Test.  Heat  50  cc.  with  2  g.  of 
phenyl  hj'drazine  hydrochloride  and  4  g.  of  sodium  acetate 
or  with  phenyl  hydrazine  acetate  (see  above). 

3.  Reaction  of  Molisch  with  increasing  dilution  in  the 
form  introduced  by  v.  Udranszky.  One  drop  of  the  urine- 
sugar  solution  B  is  diluted  with  ten  drops  (0.5  cc.)  of  w^ater; 
one  drop  of  a-naphthol  solution  and  1  cc.  of  sulphuric  acid 
are  added.  Repeat  in  the  same  way  with  the  urine  as  a 
check.  Then  dilute  both  urines  and  repeat  the  test.  As  a 
rule  normal  urine  when  diluted  five  times  still  gives  a  sug- 
gestion of  the  reaction.  If  the  reaction  appears  at  even 
greater  dilution,  then  the  amount  of  carbohydrate  is  above 
the  normal.  Since  this  excess  need  not  necessarily  be  sugar 
and  the  limit  of  the  reaction  for  the  normal  amount  of  car- 
bohydrate is  not  fixed,  the  test  is  not  to  be  regarded  as  con- 
clusive. 

4.  Reaction  with  Nylander's  Bismuth  Solution.  Boil  5 
cc.  of  urine  with  ten  drops  (0.5  cc.)  of  the  bismuth  solution  for 
some  minutes.  The  urine-sugar  solution  B  turns  black,  the 
urine  used  as  a  check  does  not.  Many  concentrated  urines, 
however,  turn  black  though  they  contain  no  sugar;  this  is 
true  also  of  those  containing  chrysophanic  acid. 


120  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

XVII.  Examination  of  Diabetic  Urine  for  Acetoacetic 
ACID,  CH3COCH2COOH. 

1,  Add  directly  to  some  of  the  urine  ferric  chloride  solu- 
tion. The  quantity  of  this  should  not  be  too  small,  since  the 
iron  chloride  is  at  first  used  up  to  form  ferric  phosphate  (gray 
precipitate):  a  red  coloration  indicates  the  presence  of 
acetoacetic  acid. 

2.  Acidify  the  urine  (about  50  cc,  sometimes  even  10  cc. 
is  sufficient)  with  dilute  sulphuric  acid,  shake  with  an  equal 
volume  of  ether,  separate  the  ether  and  agitate  it  with  a  very 
little  dilute  ferric  chloride  solution.  In  the  presence  of 
acetoacetic  acid  the  aqueous  layer  turns  violet-red.  After 
the  use  of  salicylic  acid  the  urine  gives  a  very  similar  reaction. 

XVIII.  ACETONE,  CH3COCH3. 

Clear  colorless  fluid  with  an  agreeable  odor,  miscible  in 
every  proportion  with  water,  alcohol,  and  ether,  boiling-point 
56°  to  57°.  To  250  cc.  of  urine  add  a  few  drops  of  acetone 
and  some  hydrochloric  acid,  distil  off  about  50  cc,  and  make 
the  following  tests  with  the  distillate : 

1.  The  Iodoform  Test. — Add  a  few  drops  of  sodium 
hydroxide  solution,  then  iodine  potassium  iodide  solution. 
The  fluid  at  once  becomes  turbid  and  gives  the  odor  of  iodo- 
form (CHI3).  On  standing  iodoform  is  deposited.  Examine 
under  the  microscope.  Aldehyde  also  gives  the  same  reac- 
tion. The  use  of  ammonia  instead  of  sodium  hydroxide  is 
said  to  exclude  this  confusion  (black  nitrogen  iodide  is  formed 
at  first  in  this  case,  but  gradually  disappears) :  this  test,  how- 
ever, is  less  delicate. 

2.  Legal's  Test. — Add  to  some  of  the  distillate  enough 
sodium  nitroprusside  solution  (freshly  prepared)  to  distinctly 
color  the  fluid  and  then  some  sodium  hydroxide  solution: 
the  fluid  turns  ruby-red.     If  acidified  with  acetic  acid  the 


EXAMINATION  OF  THE  URINE.  121 

color  becomes  more  violet.     Aldehyde  gives  the  same  reac- 
tion. 

3.  Gunning's  Test.— Add  to  some  of  the  distillate  a  few 
drops  of  mercuric  chloride  solution,  then  some  sodium  hydrox- 
ide solution  and  an  equal  volume  of  alcohol.  Shake  thor- 
oughly antl  filter  through  a  close  filter.  The  filtrate  must  be 
quite  clear.  Acidify  the  filtrate  faintly  with  hydrochloric 
acid  and  float  on  it  some  ammonium  sulphide  solution,  so  as 
to  form  two  layers.  At  the  surface  of  contact  a  grayish- 
black  ring  of  mercuric  sulphide  wll  appear.  The  reaction 
depends  upon  the  solubility  of  mercuric  hydroxide  in  ace- 
tone.    Aldehyde  also  possesses  this  property  (v.  Jaksch). 

The  confusion  with  aldehyde  is  not  especially  to  be  feared, 
since  this  substance  has  not  hitherto  been  found  in  urine 
and  could  only  be  present  if  the  acidified  urine  should  be 
carelessly  distilled  too  far.  Aldehyde  may  be  readily  detected 
in  the  distillate  by  the  following  reactions : 

1.  If  we  add  to  a  small  portion  some  ammoniacal  silver 
solution  (to  prepare  this  add  to  about  5  cc.  of  silver  nitrate 
solution  some  drops  of  ammonia  and  then  half  the  volume  of 
sodium  hydroxide  solution),  blackening  quickly  takes  place. 

2.  If  we  warm  a  small  portion  after  the  addition  of  some 
sodium  hydroxide  solution,  a  yellow  color  appears  (eventually 
turbidity  also)  and  a  characteristic  odor  develops  (formation 
of  aldehyde  resin). 

XIX.  Detection  of  bile-pigment. 

1.  Gmelin's  Test.  Float  some  icteric  urine  by  means  of  a 
pipette  on  a  few  cubic  centimeters  of  nitric  acid,  which  con- 
tains a  very  small  amount  of  nitrous  acid  (fuming  nitric  acid) : 
colored  rings  where  the  two  liquids  meet  and  in  the  follow- 
ing order  from  top  to  bottom:  green,  blue,  violet,  red. 

2.  Rosenbach's  Modification  of  Gmelin's  Test.  Filter  a 
quantity  of  the  urine,  partly  dry  the  filter  by  pressing  between 


122  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

drying-paper,  and  moisten  its  inner  surface  with  yellow  nitria 
acid  such  as  was  used  in  experiment  1. 

3.  Make  the  urine  alkaline  with  a  few  drops  of  sodium 
carbonate  and  then  add,  with  constant  shaking,  drop  by  drop^ 
calcium  chloride  solution  until  the  supernatant  fluid  shows 
no  perceptible  color  or  only  the  normal  urine  color. 

Filter  off  the  precipitate,  wash  well,  put  it  into  a  test-tube^, 
pour  on  some  alcohol,  and  dissolve  the  precipitate  by  adding 
hydrochloric  acid  and  shaking.  If  the  clear  solution  be  boiled 
it  turns  green  to  blue  in  the  presence  of  bile-pigment,  and  in 
the  absence  of  this  it  remains  uncolored.  Let  cool  completely 
and  add  nitric  acid.  The  green  color  changes  to  blue,  violet, 
and  red.  According  to  Hammarsten  a  mixture  of  nineteen  vol- 
umes of  hydrochloric  acid  with  one  of  nitric  acid  is  prepared. 
One  volume  of  this  mixture  is  added  to  five  to  nine  volumes 
of  alcohol  and  the  precipitate  dissolved  in  this :  green  solution 
which  gradually  turns  blue  when  bile-pigments  are  present. 

The  Gmelin  test  may  be  doubtful  or  lead  to  errors  if  the 
urine  contains  considerable  indican.  This  is  excluded  by  the 
isolation  of  the  bile-pigment  in  method  3.  Moreover,  the 
test  3  often  gives  a  positive  result  when  test  1  does  not. 

XX.  Detection  of  dissolved  Blood-pigments.^ 

Take  400  cc.  of  normal  urine.  Add  to  300  cc.  of  this  2  cc. 
of  diluted  blood  (1  :  10)  and  shake  thoroughly.  Reserve  the 
other  100  cc.  for  check  tests.  The  color  of  the  urine  to  which 
the  blood  has  been  added  would  not  lead  us  to  suspect  the 
presence  of  the  blood-pigment. 

1.  Spectroscopic  Examination  Direct.  If  the  bands  of  the 
oxyhsemoglobin  are  not  to  be  seen,  add  to  the  urine,  accord- 
ing to  L.  Lewin  and  Posner,  some  drops  of  ammonium  sulphide 
and  then  a  few  drops  of  sodium  hydroxide  solution.     The 

^  Oxyhsemoglobin  and  methaemoglobin. 


EXAMINATION  OF  THE   URINE.  123 

examination  in  the  spectroscope  then  shows  the  very  char- 
acteristic bands  of  reduced  hsematin  (haemochromogen  of 
Hoppe-Seyler), 

2.  Heller's  Test.  Make  a  portion  of  the  urine  strongly 
alkahne  with  sodium  hydroxide  solution,  heat  to  boiling,  and 
let  stand.  The  precipitate  of  phosphates,  which  collects  on 
the  bottom  of  the  test-tube,  is  colored  blood-red  by  hsematin. 
Make  a  check  experiment  with  normal  urine. 

3.  Add  to  100  cc.  of  the  urine  some  cubic  centimeters  of 
urine  containing  considerable  albumin,  heat  to  boiUng,  col- 
lect the  precipitate  on  a  filter,  and  wash.  Grind  the  precipi- 
tate in  a  mortar  -^dth  about  20  cc.  of  absolute  alcohol,  add  a 
few  drops  of  concentrated  sulphuric  acid,  heat  the  mixture  to 
boihng  in  a  flask  on  the  water-bath,  and  filter.  When  cold 
make  the  filtrate  alkaline  with  sodium  hydroxide  solution  and 
add  a  few  drops  of  ammonium  sulphide.  When  examined 
with  the  spectroscope  the  fluid  now  shows  the  absorption- 
bands  of  reduced  hsematin. 

4.  Add  to  the  urine  a  Httle  freshly  made  alcoholic  solution 
of  gum  guaiacum  till  a  permanent  turbidity  results,  then  some 
old  oil  of  turpentine,  and  shake  thoroughly.  On  allowing  to 
stand  and  shaking  repeatedly  the  mixture  or  the  oil  of  tur- 
pentine gradually  becomes  blue.  On  shaking  this  mixture 
with  ether  a  violet  color  is  imparted  to  the  ether  and  the  blue 
color  remains  in  the  aqueous  fluid.  Both  gradually  fade. 
Make  a  check  experiment  with  normal  urine.  Not  decisive 
in  the  presence  of  pus-cells.  The  latter,  according  to  Brand- 
enburg,' give  the  blue  color  even  with  the  guaiacum  tincture 
alone.    • 

XXI.  Detection  of  H^ematoporphyrin. 

1.  Thirty  to  fifty  cubic  centimeters  of  urine  containing 
hajmatoporphyrin  are  completely  precipitated  with  an  alka- 

'  Miinchoner  nicdicin.  Wochenschr.  1900.  No.  6. 


124  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

line  barium  chloride  solution  (mixture  of  equal  volumes  of  a 
cold  saturated  barium  hydroxide  solution  and  a  10  per  cent, 
barium  chloride  solution),  the  precipitate  is  washed  a  few 
times  with  water,  then  once  with  absolute  alcohol,  and  drained 
as  completely  as  possible.  Place  the  moist  precipitate  in  a 
small  mortar,  add  six  to  eight  drops  of  hydrochloric  acid,  then 
enough  absolute  alcohol  to  make  a  thin  paste,  grind  well,  let 
stand  for  some  time  or  warm  gently  on  the  water-bath,  and 
filter  through  a  dry  filter.  If  the  mixture  gives  too  small  a 
filtrate,  wash  with  some  alcohol.  It  is  advisable,  however, 
to  prepare  not  more  than  8  to  10  cc.  of  the  alcoholic  extract. 
The  coloring-matter  may  also  be  extracted  from  the  precipi- 
tate, after  it  has  been  washed  with  water  and  alcohol,  by 
repeatedly  pouring  upon  it  a  warmed  mixture  of  about  10  cc. 
of  absolute  alcohol  and  six  to  eight  drops  of  hydrochloric  acid. 
The  alcoholic  extract  is  red  and  shows  the  two  characteristic 
bands  of  hsematoporphyrin  in  acid  solution  (see  Table  of 
Absorption  Spectra,  No.  6).  If  the  solution  is  made  alkaline 
with  ammonia  it  takes  on  a  yellowish  shade  and  now  shows 
the  four  absorption-bands  of  hsematoporphyrin  in  alkaline 
solution. 

2.  Add  to  some  of  the  same  urine  some  glacial  acetic 
acid  (to  100  cc.  of  urine  5  cc.  of  glacial  acetic  acid)  and 
let  stand  for  two  days;  the  hsematoporphyrin  is  deposited 
as  a  precipitate  (Nebelthau). 

EXAMINATION   FOR  THE   INORGANIC   CONSTITUENTS. 

I.  Detection  of  Chlorides. 

Add  to  the  urine  a  few  drops  of  nitric  acid  and  then  some 
silver  nitrate  solution;  according  to  the  amount  of  chlorides 
present  in  the  urine  there  will  be  formed  either  a  white  pre- 
cipitate of  silver  chloride,  AgCl,  which  on  shaking  fonns 
white  cheesy  flakes  (normal  conduct),  or  only  a  faint  tur- 
bidity (febrile  urine). 


EXAMINATION  OF  THE   URINE.  125 

II.  Detection  of  Sulphates. 

Add  some  hydrochloric  acid  to  the  urine  and  then  a  little 
barium  chloride  solution:  white  precipitate  of  barium  sul- 
phate, BaS04  (normal  conduct),  or  only  a  faint  turbidity 
(after  the  use  or  absorption  of  considerable  carbolic  acid). 

III.  Detection  of  Ethereal  Sulphates. 

Mix  20  cc.  of  the  urine  and  20  cc.  of  the  alkahne  barium 
chloride  solution  (see  page  124)  and  filter.  Boil  the  filtrate 
with  fuming  hydrochloric  acid  (one-half  volume):  turbidity 
due  to  the  precipitation  of  barium  sulphate,  which  is  ordi- 
narily shght,  but  if  the  amount  of  ethereal  sulphates  is 
abnormally  large  (from  different  causes)  it  may  be  consider- 
able. 

IV.  Detection  of  Phosphates. 

(a)  In  general.  About  20  cc.  of  urine  are  treated  with 
acetic  acid  and  uranyl  acetate  solution:  yellowish-white 
precipitate  of  uranium  phosphate  or  uranyl  phosphate 
(U02)HP0,. 

(6)  Separate  Detection  of  Phosphoric  Acid  united  to  the 
Alkali  Metals  and  to  the  Alkali-earth  Metals.  Make  50  cc. 
of  urine  alkaUne  with  ammonia  and  after  the  mixture  has 
stood  for  some  time  filter  from  the  precipitate  of  alkali- 
earth  phosphates.  The  filtrate  contains  the  phosphoric  acid 
combined  with  the  alkali  metals,  as  may  be  shown  by  the 
addition  of  acetic  acid  and  uranyl  acetate  solution.  Dissolve 
the  precipitate,  after  it  has  been  thoroughly  washed  with 
water,  by  pouring  on  acetic  acid;  phosphoric  acid  may  also 
be  shown  to  be  present  in  this  solution  by  means  of  the  uranyl 
acetate  solution. 

V.  Detection  of  Ammonium  Salts. 

Put  25  cc.  of  the  urine,  a  few  drops  of  thymol  solution,  and 
about  25  cc.  of  milk  of  Hmc  in  the  Schlosing  apparatus  (an 


126  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

empty  desiccator  may  be  used),  and  in  the  dish  5  cc.  of  water 
containing  a  few  drops  of  hydrochloric  acid  to  absorb  the 
ammonia.  Let  stand  for  forty-eight  hours.  Show  the  pres- 
ence of  ammonia  in  the  water  in  the  usual  manner  (by  means 
of  platinum  chloride  or  Nessler's  reagent). 

VI.  Detection  of  Potassium  Iodide. 

Urine,  voided  after  the  use  of  potassium  iodide,  or  one  to 
which  potassium  iodide  (0.2  per  cent.)  has  been  added,  is 
treated  with  nitric  acid  containing  some  nitrous  acid,  a  few 
cubic  centimeters  of  chloroform  are  added  and  the  mixture 
well  shaken:  the  chloroform  becomes  colored  violet.  With 
urines  containing  considerable  indican  errors  may  arise  from 
the  formation  of  indigo  red  and  indigo  blue.  In  order  to 
exclude  these,  add  to  the  solution  a  httle  starch  paste  and 
shake  thoroughly.  At  the  surface  of  contact  of  the  chloro- 
form and  the  urine  a  ring  of  the  blue  starch-iodine  product 
will  be  formed. 

If  the  color  is  due  to  iodine  it  will  be  removed  by  the  addi- 
tion of  a  solution  of  sodium  thiosulphate,  while  the  indigo 
color  will  remain  unchanged. 

VII.  Detection  of  Potassium  Bromide. 

Ten  to  twenty  cubic  centimeters  of  urine,  voided  after 
the  use  of  potassium  bromide,  or  one  which  contains  0.2  per 
cent,  of  potassium  bromide,  are  made  alkaline  with  sodium 
carbonate,  about  3  g.  of  potassium  nitrate  added,  evaporated 
to  dryness  in  a  silver  or  platinum  dish,  and  then  heated  more 
strongly  till  the  mass  melts  and  becomes  perfectly  white. 
After  cooUng,  dissolve  the  fused  mass  in  water,  acidify  strongly 
with  hydrochloric  acid  (not  sulphuric  acid),  add  some  fresh 
chlorine-water,  and  shake  with  chloroform:  the  chloroform 
becomes  colored  yellow. 


EXAMINATION  OF   THE   URINE.  127  . 

VIII.  Detection  of  Mercury. 

Method  of  Fiirbringer.  Add  to  500  cc.  of  urine  1  to  2  cc. 
of  mercuric  chloride  solution  (0.1  per  cent.)  and  10  cc.  of 
hydrochloric  acid.  Heat  to  60-80°  and  digest  for  about  a 
quarter  of  an  hour  with  0.25  to  0.5  g.  of  very  fine  brass  shav- 
ings, pour  off  the  urine,  rinse  the  brass  shavings  repeatedly 
with  warm  water,  then  with  alcohol,  and  finally  two  or  three 
times  with  ether.  Then  place  the  brass  shavings,  twisted 
into  a  roll,  in  a  glass  tube  closed  at  one  end,  about  10  cm.  long 
and  8  mm.  wide,  and  draw  out  the  tube  at  some  distance  from 
the  brass  shavings  to  a  fine  point.  Heat  the  brass  shavings 
gently  from  the  bottom  upwards:  a  sublimate  forms  which 
should  be  driven  up  to  the  capillary.  This  subUmate  is  by 
no  means  all  mercury,  but  contains  zinc  oxide,  and  some- 
times drops  of  water  also  condense.  To  detect  the  mercury 
it  must  be  converted  into  the  iodide.  This  is  best  done  in 
the  following  manner:  Cut  off  the  imder  part  of  the  tube, 
thus  removing  the  roll  of  brass  shavings,  place  in  the  upper 
part  of  the  tube  a  grain  of  iodine,  convert  this  into  vapor  by 
gently  warming,  and  blow,  with  the  aid  of  a  glass  tube,  gently 
into  the  upper  opening  of  the  tube.  The  iodine  vapor  is  thus 
forced  over  the  mercury  and  changes  this  into  mercuric 
iodide.  Instead  of  this  some  iodine  may  be  placed  in  the 
under  part  of  the  tube,  which  is  then  fused  together  and  the 
iodine  volatiUzed  by  heating  gently.  Examine  the  tube  with 
a  magnifying-glass  or  under  the  microscope :  especially  char- 
acteristic is  the  conversion  of  the  yellow  mercuric  iodide,  if 
it  is  present,  into  the  red  variety  by  touching  it  with  a  plati- 
num wire,  as  well  as  the  volatility  of  the  mercuric  iodide  and 
its  crystal  form. 


CHAPTER  XI. 

EXAMINATION  OF  URINARY  CALCULI. 

Heat  a  portion  of  the  finely  powdered  calculus  on  plati- 
num-foil; if  it  burns  completely  or  leaves  only  a  very  small 
quantity  of  ash,  then  it  consists  of  uric  acid,  ammonium 
urate,  cystine,  or  xanthine.  If  it  does  not  burn  completely 
it  may  contain  uric  acid  or  urates,  calcium  phophate,  and 
magnesium  phosphate,  or  ammonium  magnesium  phosphate 
and  calcium  oxalate.  The  method  to  be  pursued  in  the 
analysis  is  based  on  this  difference. 

I.  THE  POWDERED  CALCULUS  BURNS  COMPLETELY. 

Digest  the  powder  by  warming  gently  with  dilute  hydro- 
chloric acid  (1  :  2). 

{a)  The  Powder  Dissolves  Completely  or  Almost  Com- 
pletely.    The  calculus  consists  of  cystine  or  xanthine. 

To  test  for  cystine  digest  a  small  portion  of  the  powder 
with  ammonia,  filter,  let  the  extract  evaporate  on  a  watch- 
glass,  and  examine  the  residue  under  the  microscope :  cystine 
forms  hexagonal  tablets.  Cystine  calculi  are  usually  small, 
have  a  yellow  color  and  a  smooth  surface. 

To  test  for  xanthine  make  the  so-called  xanthine  test  with 
nitric  acid  and  sodium  hydroxide  solution  (see  chapter  on 
Muscular  Tissue,  page  29). 

(6)  The  Powder  Does  Not  Dissolve  Completely.  Filter  and 
wash  the  residue. 

128 


EXAMINATION  OF  URINARY  CALCULI.  129 

1.  Residue:  Uric  acid.  Confirm  by  making  the  murexide 
test  (page  101).  Uric  acid  calculi  vary  in  size,  are  quite  hard, 
and  are  usually  colored  reddish  yellow  or  brown. 

2.  Filtrate:  may  contain  ammonium  chloride.  To  test 
for  ammonia  warm  some  of  the  filtrate  with  sodium  carbonate 
solution:  ammonia  is  evolved  and  may  be  detected  by  its 
odor,  alkaUne  reaction,  etc. 

II.  The  Powder  Turns  Black  but  Does  Not*  Burn. 

A  slight  blackening  always  results  when  the  calculi  are 
heated,  due  to  the  presence  of  organic  matter.  A  small  por- 
tion of  the  finely  powdered  calculus  is  digested  by  warming 
with  dilute  hydrochloric  acid  (1  :  2):  effervescence  indicates 
the  presence  of  carbonates. 

(a)  Complete  Solution.    Uric  acid  is  not  present. 

(6)  Incomplete  Solution.  The  residue  may  contain  uric 
acid,  proteids,  epithelium,  etc.  The  general  appearance  or 
a  microscopical  examination  usually  enables  us  to  decide 
what  is  present.  The  presence  of  the  uric  acid  may  be 
readily  confirmed  by  the  murexide  test. 

In  any  case  the  solution  is  to  be  further  investigated. 
Warm  a  small  portion  of  the  filtered  solution  with  an  excess 
of  sodium  carbonate  solution  and  test  for  ammonia  (see 
above).  Dilute  the  main  quantity  of  the  liquid  with  water, 
filter,  make  faintly  alkaline  with  ammonia,  cool  the  fluid  in 
case  it  has  become  hot  from  the  addition  of  the  ammonia,  and 
acidify  with  acetic  acid.  Either  an  approximately  clear  solu- 
tion results  or  a  turbid  one,  which  gradually  deposits  a  white 
pulverulent  precipitate. 

The  yellowish-white  flakes  which  are  seen  in  the  approxi- 
mately clear  solution  consist  of  ferric  phosphate.  Prove  this 
by  filtering  and  dissolving  the  washed  precipitate  in  hydro- 
chloric acid:  the  solution  is  colored  blue  on  the  addition  of 
potassium  ferrocyanide. 


130  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

The  white  insoluble  precipitate  is  calcium  oxalate.  Prove 
this  by  a  microscopical  examination.  Filter;  if  the  quantity 
is  not  too  small,  wash,  dry,  and  heat  to  red  heat  on  platinimi- 
foil.  The  calcium  oxalate  burns  to  calcium  carbonate  and 
oxide.  Therefore  the  residue,  when  moistened  with  a  drop 
of  water,  shows  a  strong  alkaUne  reaction  and  dissolves  in 
hydrochloric  acid  with  effervescence.  The  filtrate  from  the 
iron  phosphate  or  the  calcium  oxalate  may  contain  phos- 
phoric acid,  calcium,  magnesium. 

1.  To  a  portion  of  the  filtrate  add  some  uranyl  acetate 
solution.  A  yellowish-white  precipitate  of  uranyl  phosphate 
indicates  the  presence  of  phosphoric  acid. 

2.  To  the  remainder  of  the  filtrate  add  ammonium  oxalate: 
a  white  precipitate  indicates  calcium.  Heat,  then  filter  from 
the  precipitate,  and  make  the  filtrate  alkahne  with  ammonia: 
a  crystalHne  precipitate  of  ammonium  magnesium  phosphate 
indicates  the  presence  of  magnesium. 

Test  for  ammonia  by  warming  with  sodium  carbonate 
solution  that  part  of  the  original  hydrochloric  acid  solution 
which  was  put  aside. 


CHAPTER  XII. 
EXAMINATION  OF  THE  LIVER. 

I.  Preparation  and  Reactions  of  Glycogen. 
II.  Detection  of  Sugar. 
III.  Preparation  of  the  Xanthine  Bases  of  the  Liver. 

I.  Preparation  of  Glycogen. 

Introduce,  by  means  of  a  tube,  into  the  stomach  of  a  well- 
fed  rabbit,  on  the  day  previous  to  killing  it,  as  well  as  five  to 
six  hours  before  death,  10  to  15  g.  of  glucose  or  cane-sugar 
dissolved  in  water.  After  killing  the  rabbit  remove  the  liver. 
Reserve  about  10  g.  of  this  for  the  experiment  below  'detec- 
tion of  sugar),  and  chop  up  the  rest  into  very  small  pieces. 
Then  heat  to  vigorous  boiling  with  ten  times  its  weight  of 
water,  adding  a  trace  of  acetic  acid  in  order  to  facihtate  the 
precipitation  of  the  proteids.  Filter  the  extract,  which  shows 
a  marked  opalescence,  through  muslin,  press  the  residue 
thoroughly,  grind  it  in  a  mortar,  and  boil  again  with  water, 
filter,  and  press  out  the  residue  as  before.  Evaporate  the 
united  extracts  to  about  100  to  150  cc,  acidify  with  hydro- 
chloric acid,  and  add  Briicke's  solution  *  (potassium  mercuric 
iodide),  then  alternately  a  few  drops  of  hydrochloric  acid  and 
Briicke's  solution  until  the  precipitation  is  complete.  The 
addition  of  the  Briicke's  solution  is  for  the  purpose  of  pre- 

'  Made  as  follows:  To  a  hot  5  to  10  per  cent,  solution  of  potassium 
iodide  add  with  constant  stirring  mercuric  iodide  until  a  portion  remains 
undissolved,  let  cool,  and  then  filter. 

131 


132  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

cipitating  the  proteids  still  present  in  the  solution  and  also 
the  gelatine  formed  on  boiling.  Now  filter,  wash  once  with 
water,  add  double  the  volume  of  90  per  cent,  alcohol,  and  stir 
thoroughly.  After  the  precipitate  has  settled  completely 
filter  and  wash,  first  with  a  mixture  of  two  volumes  of  alco- 
hol and  one  volume  of  water,  then  with  absolute  alcohol,  and 
finally  with  ether;  or,  in  case  it  is  somewhat  voluminous,  it 
is  better  to  remove  it  from  the  filter,  grind  with  absolute 
alcohol,  let  stand  some  time  with  this,  filter,  press  out  the 
alcohol,  and  treat  in  the  same  manner  with  ether.  Finally 
the  glycogen  is  freed  from  the  adhering  ether  by  pressing  and 
by  grinding  in  a  mortar.^  The  method  of  preparation  of 
S.  Frankel  ^  is  also  very  convenient:  Grind  the  liver,  without 
heating  it,  with  2.5  times  the  quantity  of  a  2  to  4  per  cent, 
solution  of  trichloracetic  acid,  filter,  wash  with  some  of  the 
trichloracetic  acid  solution,  and  precipitate  with  alcohol,  etc. 
The  trichloracetic  acid  has  the  property  of  coagulating  the 
proteids  and  completely  precipitating  them. 

Thus  prepared  glycogen,  CqH^JJ^  (according  to  Huppert 
6(C6Hio05)  +  H20),  is  a  chalky-white  fine  powder  in  which 
hard  transparent  pieces  resembling  gum  arable  may  be  pres- 
ent in  case  of  insufficient  dehydration;  it  dissolves  readily, 
though  somewhat  slowly,  in  water,  always  forming  an  opales- 
cent solution,  which  is  extremely  strongly  dextrorotatory 
(according  to  E.  Kiilz  ckj  is  +211°,  according  to  Huppert  a^  is 
-1-196.63°).  On  boihng  with  acids  it  forms  glycogen-dextrin 
and  then  glucose;  when  treated  with  saliva  or  pancreas 
extract  it  is  converted  into  glycogen-dextrin  and  maltose. 
It  forms,  Hke  other  carbohydrates,  oxalic  acid  when  boiled 
with  nitric  acid.  It  is  distinguished  by  its  characteristic  con- 
duct towards  iodine  solution. 

^  For  a  better  method  for  the  preparation  of  glycogen  see  Salkowski, 
Zeit.  f.  Physiol.  Chem.  36  (257).     See  also  page  131  of  this  book.— O. 
2  Pfluger's  Archiv,  52,  125. 


EXAMINATION  OF  THE  LIVER.  133 

Reactions  of  Glycogen. 

1.  Heat  a  small  portion  of  the  substance  on  platinum-foil 
until  all  the  carbon  is  burned:  only  a  very  small  amount  of 
ash  should  remain. 

2.  Dissolve  0.25  g.  by  warming  with  50  cc.  of  water  or 
0.5  g.  in  100  cc. 

(a)  Determination  of  the  rotation:  If  the  dextrorotation  is 
not  distinctly  apparent,  add  some  sodium  hydroxide  solution. 

(6)  Add  to  a  small  portion  of  the  solution  a  very  small 
quantity  of  iodine  potassium  iodide  solution  saturated  with 
sodium  chloride.  The  solution  turns  reddish-brown;  con- 
tinue to  add  the  iodine  as  long  as  the  intensity  of  the  color 
perceptibly  increases,  then  divide  the  mixture  into  two  parts. 
Heat  one  part  gently:  the  color  disappears,  but  returns  on 
cooUng.  The  addition  of  sodium  hydroxide  decolorizes  the 
solution  at  once,  sodium  carbonate  acts  more  slowly  (com- 
bination with  the  iodine),  acids  gradually  decolorize  the  solu- 
tion (forming  sugar) . 

According  to  Hoppe-Seyler,  in  order  to  determine  the 
presence  of  glycogen  in  neutral  solutions,  place  equal  por- 
tions of  dilute  iodine  solution  in  two  test-tubes  of  the  same 
diameter,  to  one  add  some  of  the  solution  to  be  tested,  to  the 
other  the  same  quantity  of  water,  and  compare  the  colors. 

(c)  Dissolve  some  commercial  peptone  in  a  few  cubic 
centimeters  of  the  glycogen  solution  and  make  the  test  with 
iodine:  the  reaction  only  appears  after  the  addition  of 
considerable  iodine  and  may  not  appear  at  all  when  the 
quantity  of  the  peptone  exceeds  that  of  the  glycogen.  The 
color  formed  frequently  disappears  on  standing  from  the 
gradual  combination  of  the  iodine.  Impure,  dilute  solutions 
of  glycogen,  therefore,  give  a  poor  iodine  reaction. 

(d)  Boil  a  few  cubic  centimeters  of  the  glycogen  solution 
a  short  time  with  about  one-third  of  its  volume  of  hydrochloric 


134  PHYSIOLOGICAL   AND  PATHOLOGICAL  CHEMISTRY. 

acid:  the  opalescence  disappears  owing  to  the  conversion  of 
the  glycogen  into  glycogen-dextrin  and  glucose.  Neutralize 
the  cold  mixture  with  sodium  hydroxide  or  make  it  faintly 
alkaUne,  add  a  few  drops  of  Fehling's  solution  and  heat:  pre- 
cipitation of  red  cuprous  oxide. 

(e)  Digest  a  few  cubic  centimeters  of  the  glycogen  solu- 
tion with  about  1  cc.  of  saliva  at  40°:  in  a  very  short  time 
the  opalescence  disappears  and  the  solution  no  longer  gives 
the  iodine  reaction;  if  the  digestion  be  continued  one  to  two 
hours  the  solution  will  give  a  marked  reaction  for  sugar 
(formation  of  maltose). 

(f)  Add  to  a  few  cubic  centimeters  of  the  solution  a  drop 
or  two  of  lead  acetate  and  then  pass  in  hydrogen  sulphide: 
deep-black  fluid,  from  which  no  lead  sulphide  separates  and 
which  is  not  changed  by  filtering;  glycogen,  like  gelatin,  has 
the  property  of  holding  fine  precipitates  in  suspension,  though 
not  to  such  an  extent. 

II.   DETECTION  OF   SUGAR. 

That  part  of  the  Hver  which  was  reserved  for  showing  the 
presence  of  sugar  is  kept  for  twenty-four  hours  and  is  then 
chopped  up.  Heat  it  to  boihng  with  ten  times  its  weight  of 
water,  adding  a  trace  of  acetic  acid  to  precipitate  the  pro- 
teids,  filter,  evaporate  to  about  one-fifth  of  its  volume,  filter 
again  and  use  the  solution  for  the  reactions  for  the  detection 
of  glucose  (see  chapter  on  Urine,  page  117).  Trommer's  test, 
the  fermentation  test,  and  the  phenylhydrazine  test  are 
sufficient. 

III.  Preparation  of  the  Xanthine  bases. 
Chop  up  finely  250  g.  of  beef-  or  calf's  Hver,  put  it  into  a  3.5 
to  4  Hter  stoppered  bottle  with  2.5  hters  of  chlorof orm- water, ^ 
then  add  about  2.5  cc.  more  of  chloroform,  shake  vigorously 

^  Made  by  shaking  vigorously  2.5  liters  of  water  with  12.5  cc.  of  chlo- 
roform in  a  glass-stoppered  bottle  till  the  chloroform  has  dissolved. 


EXAMINATION  OF  THE  LIVER.  135 

several  times,  then  digest  two  to  three  days  in  an  air-bath  at 
40°.  The  digestion  \Aith  chloroform-water  effects  two 
objects:  (1)  it  causes  the  complete  cleavage  of  the  nucleins; 
and  (2)  it  removes  substances,  always  present  in  the  extracts 
of  organs,  which  disturb  or  entirely  prevent  the  precipitation 
of  the  xantliine  bases  by  silver  nitrate.  The  first  object 
can  only  be  attained  otherwise  by  boiling  with  dilute  acids 
(Kossel).  Wliether  the  second  may  also  be  completely 
effected  by  dilute  acids  alone  has  not  been  determined  as  yet 
with  certainty,  though  it  is  probable.  At  any  rate,  the 
digestion  with  chloroform-water  has  the  advantage  of  great 
convenience. 

After  the  cUgestion  heat  the  mixture  to  boihng  in  a  large 
enameled  iron  dish  or  agate-ware  pan  and  continue  the  boil- 
ing, after  acidifjing  faintly  with  acetic  acid,  until  the  albumin 
has  been  completely  precipitated.  Then  filter  and  evaporate 
further  to  a  volume  of  800  to  1000  cc.  Make  the  solution 
moderately  alkahne  with  ammonia,  filter  from  the  shght 
precipitate  formed,  and  precipitate  completely  with  a  3  per 
cent,  ammoniacal  solution  of  silver  nitrate:  a  gelatinous 
precipitate  of  the  silver  compounds  of  the  xanthine  bases  is 
formed.  Care  must  be  taken  that  no  silver  chloride  precipi- 
tates. If  this  should  happen,  more  ammonia  must  be  added. 
Add  the  silver  nitrate  solution  imtil  a  small  portion  of 
the  filtrate,  after  acidifying  with  nitric  acid  and  adding 
some  hydrochloric  acid,  gives  a  milky  turbidity.  Filter  off 
the  precipitate,  wash  well,  place  it  in  a  flask  and  dissolve  it 
while  still  moist  in  hot  nitric  acid  of  1.1  specific  gravity 
(equal  volumes  of  nitric  acid,  specific  gravity  1.2,  and  water) 
to  which  some  urea  has  been  added.  The  solution  must  be 
almost  clear.  Filter  hot  and  let  stand  twenty-four  hours. 
Guanine-,  adenine-  and  hypoxanthine-silver  nitrate  crystal- 
lize out,  while  xanthine-silver  nitrate  remains  in  solution. 
Filter  and  wash  the  precipitate. 


136  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

(a)  Make  the  filtrate  alkaline  with  ammonia:  precipitate 
of  xanthine-silver.  For  further  treatment  see  chapter  on 
Muscular  Tissue,  page  25. 

(&)  Suspend  the  precipitate  of  guanine-,  adenine-  and  hypo- 
xanthine-silver  nitrate  in  water  and  decompose  by  passing  in 
hydrogen  sulphide,  filter  from  the  silver  sulphide,  evaporate 
to  a  small  volume,  make  alkaline  with  ammonia,  and  let  stand. 
Guanine  crystallizes  out,  while  hypoxanthine  and  adenine 
(besides  ammonium  nitrate)  remain  in  solution.  Filter, 
wash  a  few  times,  and  use  the  guanine  for  the  reactions. 

Guanine,  C5H5N5O  (amino-hypoxanthine,  G5H3N40(NH2), 
is  insoluble  in  water,  soluble  in  sodium  and  potassium  hydrox- 
ide solutions  and  also  in  acids  forming  salts.  It  is  distin- 
guished from  the  other  xanthine  bases  by  being  almost  insolu- 
ble in  ammonia.  Of  all  the  xanthine  bases  it  gives  the  strong- 
est so-called  xanthine  reaction  and,  hke  xanthine,  gives  a 
reaction  with  hypochlorites.  Like  hypoxanthine  and  adenine 
the  compound  with  silver  nitrate  is  insoluble  in  dilute  nitric 
acid. 

Reactions  of  Guanine. 

1.  Try  the  xanthine  reaction  (see  chapter  on  Muscular 
Tissue,  page  29).  The  residue  left  after  evaporating  the 
nitric  acid  solution  turns  intensely  dark  red,  even  bluish  red 
on  moistening  with  sodium  hydroxide  solution. 

2.  Dissolve  a  small  portion  in  hydrochloric  acid  and  add 
a  little  saturated  aqueous  solution  of  picric  acid:  a  crys- 
talline precipitate  gradually  forms. 

3.  Mix  in  a  watch-glass  some  sodium  hydroxide  solution 
with  a  little  chloride  of  Hme  and  put  into  the  mixture  a  grain 
of  guanine;  around  this  there  will  be  formed  a  dark-green 
ring.  This  color  soon  turns  to  brown  and  then  gradually 
disappears.  Xanthine  gives  the  same  reaction  (originally 
given  by  Hoppe-Seyler  for  xanthine). 


CHAPTER  XIII. 

EXAMINATION  OF  BONE. 

Pour  10  cc.  of  water  upon  some  pieces  of  hollow  bone 
(about  3  g.)  in  a  beaker,  add  10  cc.  of  hydrochloric  acid 
(evolution  of  carbon  dioxide  follows  the  addition  of  the 
acid),  and  let  stand  at  room  temperature  for  twenty-four 
hours.  The  dilute  hydrochloric  acid  dissolves  the  inorganic 
constituents  of  the  bone  and  leaves  the  ossein,  which  retains 
the  shape  of  the  original  bone,  undissolved. 

I.  Ossein  and  Gelatin  (Glutin). 

Pour  off  the  hydrochloric  acid  solution  and  preserve  for 
further  investigation  (under  III).  Wash  the  ossein  several 
times  with  water,  then  let  it  lie  in  water  containing  a  few 
drops  of  sodium  carbonate  solution  and  again  wash  with 
water.  Place  it  in  a  beaker  with  a  small  quantity  of  water, 
heat  this  to  boiling,  and  continue  the  boihng  until  the  pieces 
of  ossein  have  for  the  most  part  dissolved  (five  to  ten  minutes)  K 
NeutraUze  or  make  the  solution  faintly  alkaline  with  sodium 
carbonate  solution,  decant  into  a  test-tube,  and  place  this  in 
cold  water.  After  some  time  the  solution  will  form  a  more 
or  less  firm  jelly  of  bone-gelatin  (also  called  glutin).  The 
ossein  is  converted  into  gelatin  or  glutin  by  boihng  with 
water. 


'  Frequently  there  remains  an  inner  part  of  the  bone,  which  was  not 
attacked  by  the  acid. 

137 


138  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

II.  Conduct  of  Gelatin  (Glutin). 

Use  a  solution  of  commercial  gelatin  (the  best  white  gel- 
atin) for  the  following  experiments. 

About  5  g.  of  gelatin  are  covered  with  water  in  an  evapo- 
rating-dish  and  allowed  to  stand.  On  the  next  day  or  after 
several  hours  it  will  appear  much  swollen,  but  not  dissolved. 
The  supernatant  fluid  is  poured  off,  40  cc.  of  water  added,  the 
mixture  heated  on  the  water-bath  until  the  gelatin  is  dis- 
solved, and  then  cooled:  very  soon  a  tolerably  firm  jelly  will 
be  obtained.  To  this  add  190  cc.  more  of  water  and  warm 
again.  The  solution  thus  obtained,  about  2  per  cent.,  is  to 
be  used  for  the  following  reactions  after  it  has  cooled  some- 
what : 

1.  Portions  of  the  solution  are  treated  in  test-tubes 
with  (a)  tannin  solution,  and  also  with  (6)  hydrochloric  and 
phosphotungstic  acids:  voluminous  precipitates.  General 
conduct  of  all  proteids,  their  immediate  derivatives  (albu- 
moses  and  peptones),  and  also  of  the  albuminoids. 

2.  Boihng  the  solution  produces  no  precipitate  even  when 
acetic  acid  is  added. 

3.  The  addition  of  acetic  acid  and  potassium  ferrocyanide 
produces  no  precipitate  (distinction  from  albumin  and  albu- 
moses) ;  under  certain  conditions,  however,  a  precipitate  may 
be  formed  (Morner).^ 

4.  The  addition  of  mercuric  chloride  gives  no  precipitate 
(distinction  from  albumoses  and  peptone). 

5.  Boiling  after  the  addition  of  one-third  of  its  volume  of 
nitric  acid  produces  only  a  very  faint  yellow  color;  gelatin 
yields  only  extremely  little  so-called  xanthoproteic  acid,  as 
the  aromatic  group  is  almost  entirely  lacking  in  the  molecule, 
and  the  phenol  or  tyrosine  group  especially  is  absent. 

6.  The  addition  of  sodium  hydroxide  and  some  copper 

1  Zeitschr.  f .  physiol.  Chem.  28,  489. 


EXAMINATION  OF  BONE.  139 

sulphate  solution  gives  a  blue-violet  color,  which,  however, 
never  shows  a  purple-red  shade  (distinction  from  peptone); 
on  heating  to  boiling  the  color  becomes  more  red,  if  but  Httle 
copper  sulphate  solution  has  been  added;  if  much  copper 
sulphate  has  been  added,  boihng  produces  no  perceptible 
color  change. 

7.  Boihng  with  Millon's  reagent  produces  only  a  faint  rose 
or  red  coloration  (it  is  advisable  to  first  heat  the  gelatin  solu- 
tion to  boiling,  add  a  few  drops  of  Millon's  reagent,  and  then 
heat  again).  Distinction  from  albumin,  due  to  the  absence 
of  the  tyrosine  group  in  the  molecule.  The  slight  red  color- 
ation is  to  be  attributed  to  admixture  with  albumoses  or 
peptone.^ 

8.  The  addition  of  bromine-water  produces  a  voluminous 
yellow  precipitate  of  a  viscous  sticky  nature. 

Gelatin  possesses  in  the  highest  degree  the  property  of 
holcHng  many  precipitates  in  the  state  of  the  finest  suspen- 
sion, so  that  they  pass  through  all  filters,  or  it  may  even 
prevent  entirely  the  formation  of  precipitates. 

(a)  Add  to  a  portion  of  the  gelatin  solution  in  a  test-tube 
a  drop  or  two  of  basic  lead  acetate:  the  solution  remains 
unchanged  (gelatin  is  not  precipitated  by  metalhc  salts  in 
general).  Now  dilute  the  mixture  to  about  30  cc.  and  pass 
in  hydrogen  sulphide:  there  results  a  brownish-black  fluid, 
which  passes  through  filter-paper  unchanged;  dilute  a  por- 
tion of  this  with  water:  a  clear  light-brown  solution  results 
from  which  no  lead  sulphide  separates. 

(6)  Dissolve  a  very  small  quantity  of  hypoxanthine  in  a 
few  cubic  centimeters  of  dilute  ammonia,  divide  the  solution 
into  two  approximately  equal  parts,  and  add  to  the  one 
(1)  double  the  volume  of  water,  to  the  other  (2)  double  the 
volume  of  the  gelatin  solution.     To  both  portions  add  silver 

'  According  to  Van  Name  pure  gelatin  gives  the  Millon's  reaction  sec 
Jour,  of  Expr.  Med.  1897,  11,  117.— O. 


140  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

nitrate  solution:  (1)  jdelds  a  flocculent  precipitate  of  hypo- 
xanthine-silver,  (2)  does  not,  the  solution  only  becomes 
faintly  opalescent.  The  gelatin  prevents  the  precipitation  of 
the  hypoxanthine-silver  completely. 

III.  The  Mineral  Constituents  of  Bone. 

Make  half  of  the  hydrochloric  acid  solution  alkaline  with 
ammonia,  then  acidify  with  acetic  acid:  the  precipitate 
formed  dissolves  on  the  addition  of  ammonia;  leaving  a  slight 
residue  of  ferric  phosphate,  which  presxmiably  comes  from 
the  blood  contained  in  the  bone  (at  least  in  part  from  this). 
Filter  and  use  a  small  part  of  the  filtrate  to  test  for  phos- 
phoric acid,  the  greater  part  to  test  for  calcium  and  magne- 
sium. 

(a)  The  flocculent  precipitate  is  washed  and  dissolved  in 
a  few  cubic  centimeters  of  dilute  hydrochloric  acid.  Test  for 
iron  in  the  solution  with  potassium  ferrocyanide,  and  for 
phosphoric  acid  with  ammonium  molybdate. 

(b)  Filtrate  from  the  ferric  phosphate. 

1.  Test  for  phosphoric  acid  by  adding  uranyl  acetate 
solution:  yellowish-white  precipitate  of  uranyl  phosphate 
(U02)HP0,. 

2.  Precipitate  the  calciimi  as  calcium  oxalate,  CaC204+H20, 
by  the  addition  of  sufficient  ammonium  oxalate.  To  the 
clear  filtrate  (made  clear  by  warming  and  pouring  repeatedly 
through  the  filter),  which  must  remain  clear  on  the  further 
addition  of  ammonium  oxalate,  add  ammonia  to  alkaline 
reaction:  a  crystalhne  precipitate  of  ammonium  magnesium 
phosphate,  MgNH4P04+6H20,  separates  after  some  minutes. 

The  detection  of  the  mineral  constituents  of  bone  may 
also  be  made  with  the  bone-ash.  For  this  purpose  0.5  to  1  g. 
of  the  bone-ash  is  sufficient.  The  carbonates  are  more  easily 
recognized  by  this  method.  The  course  of  the  examinatioii 
is  the  same. 


CHAPTER  XIV. 
EXAMINATION  OF  ADIPOSE  TISSUE. 

I.  Separation  of  Fat  and  Connective  Tissue. 
II.  Decomposition  of  Fat  into  Fatty  Acids  and  Glycerin. 

I.  Separation  of  fat  and  Connective  Tissue. 

Cut  into  fine  pieces  with  a  knife  or  a  pair  of  shears  10  g. 
of  adipose  tissue/  grind  as  finely  as  possible  in  a  mortar, 
place  in  a  flask,  and  heat  to  boiling  on  a  water-bath  with 
40  cc.  of  absolute  alcohol.  The  fat  dissolves,  leaving  the 
connective  tissue  behind.  Filter,  wash,  first  with  alcohol, 
then  once  or  twice  with  ether,  press  the  residue  on  the  filter 
between  filter-paper,  and  allow  the  ether  still  adhering  to 
evaporate  by  letting  it  He  in  the  air:  fibrous  mass  consisting 
of  fat-cells  and  connective  tissue.  Show  the  presence  of  albu- 
min in  the  residue  by  heating  with  nitric  acid  and  then  add- 
ing sodium  hydroxide  solution  (xanthoproteic  reaction),  and 
also  by  boihng  some  of  the  substance  with  water  and  adding 
a  few  drops  of  Millon's  reagent.  The  conversion  of  the  con- 
nective tissue  into  gelatin  cannot  be  brought  about  by  simply 
boiUng  with  water  as  in  the  case  of  ossein.  A  higher  tem- 
perature (boiling  under  pressure)  or  very  long-continued  boil- 
ing is  required  to  accomphsh  this. 

•  Preferably  unsmoked  hog-fat. 

141 


142  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

On  cautious  evaporation  on  the  water-bath,  the  ethereal- 
alcoholic  solution  yields  fat,  which  slowly  solidifies. 

Reactions  of  Fat. 

1.  Rub  a  small  portion  of  the  fat  on  paper  (not  filter- 
paper).    The  paper  becomes  transparent. 

2.  Add  to  a  few  cubic  centimeters  of  alcohol  one  to  two 
drops  of  very  dilute  sodium  hydroxide  solution  (about  tenth- 
normal caustic  soda  solution  containing  0.4  per  cent  of  NaOH), 
then  add  enough  rosohc  acid  solution  or  phenolphthalein 
solution  to  make  the  fluid  intensely  red.  At  the  same  time 
dissolve  a  httle  fat  (one  drop  or  a  piece  the  size  of  a  pea) 
in  a  few  cubic  centimeters  of  ether  and  pour  the  ethereal 
solution  of  the  fat  into  the  solution  of  the  indicator.  The 
solution  does  not  change  its  red  color,  the  fat  reacts  neutral. 

3.  Grind  in  a  mortar  a  small  quantity  (one  drop  or  a  piece 
the  size  of  a  pea)  with  some  powdered  potassium  bisulphate 
(monopotassium  sulphate)  and  heat  the  mixture  in  a  dry 
test-tube:  penetrating  odor  (caution!)  of  acrolein  (acryhc 
aldehyde,  CH2  =  CH— CHO).  A  strip  of  filter-paper  moist- 
ened with  an  ammoniacal  silver  nitrate  solution  (see  Acetone, 
page  121)  turns  black  imanediately  (reduction  to  silver)  when 
placed  in  the  upper  part  of  the  tube.  The  fat  is  here  decom- 
posed and  the  glycerin  converted  into  acrolein  by  the  elimi- 
nation of  water. 

4.  Warm  a  small  portion  of  the  fat  in  a  test-tube  with 
sodium  carbonate  solution:  the  fat  forms  a  temporary  emul- 
sion, but  does  not  dissolve;  saponification  does  not  take  place. 
Sodium  hydroxide  solution  also  does  not  saponify  fat  at  room 
temperatures. 

II.   DECOMPOSITION  OF  FAT,   SAPONIFICATION. 

When  heated  with  caustic  potash  or  caustic  soda  (very 
readily  in  alcoholic  solution)  the  fats  are  decomposed  with 


EXAMINATION  OF  ADIPOSE  TISSUE.  143 

the  assumption  of  the  elements  of  water,  into  fatty  acids, 
which  combine  \\ith  the  alkaUes  to  form  soaps  and  glycerin; 
for  example, 

CsiH^^Oe  +  3H30  =  3(C,eH3303)  +  C3HSO3. 

Tripalmitin  Water         Palmitic  Acid  Glycerin 

Process  of  Saponification. 

Weigh  off  about  15  g.  of  caustic  potash  in  an  evaporating- 
dish,  add  10  cc.  of  water,  and  heat  on  the  water-bath  until 
the  caustic  potash  has  dissolved.  At  the  same  time  make  up 
100  cc.  of  90  per  cent,  (volume  per  cent.)  alcohol  in  a  measur- 
ing-cyUnder.  Pour  the  caustic  potash  solution  into  a  400-cc. 
flask  and  rinse  out  the  dish  with  a  part  of  the  alcohol.  Then 
weigh  off  50  g.  of  lard  in  an  evaporating-dish,  place  the  dish 
on  the  water-bath,  heat  till  the  fat  is  completely  melted,  pour 
the  melted  fat  into  the  same  flask,  wash  out  the  fat  remaining 
in  the  dish  by  heating  with  portions  of  the  alcohol  on  the 
water-bath,  and  finally  pour  the  rest  of  the  alcohol  into  the 
flask.  Place  the  flask  on  a  hot-water  bath,  heat,  and  cau- 
tiously shake  the  contents  thoroughly  as  soon  as  the  alcohol 
begins  to  boil.  Saponification  takes  place  very  quickly, 
almost  immediately.'  In  order  to  determine  with  certainty 
whether  the  saponification  is  completed,  pour  a  small  quan- 
tity of  the  alcohohc  fluid  into  a  httle  distilled  water:  the  solu- 
tion must  be  clear;  it  should  contain  no  unsaponified  fat  in 
the  form  of  drops  of  oil.  The  solution  will  then  contain  soap 
and  glycerin  besides  the  excess  of  caustic  potash  and  the 
alcohol. 

Separation  of  the  Fatty  Acids  and  the  Glycerin. 
Pour  the  contents  of  the  flask  gradually  and  with  con- 
stant stirring  into  hot  dilute  sulphuric  acid  contained  in  a 

»  If  we  heat  the  alcohohc  solutions  of  the  fat  and  caustic  potash  to 
boiling  separately  and  pour  the  two  solutions  together,  saponification 
-does  take  place  at  once  on  shaking. 


144  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

beaker.  The  sulphuric  acid  must  be  somewhat  more  than 
equivalent  to  the  caustic  potash  used  (12  g.  of  concentrated 
sulphuric  acid  poured  into  250  cc.  of  water  or  60  cc.  of  20  per 
cent,  sulphuric  acid  ^  and  200  cc.  of  water).  The  fatty  acids, 
separate  as  an  oily  layer.  When  all  of  the  soap  solution  has 
been  added,  let  cool  or  cool  in  water,  break  up  the  layer  of 
fatty  acids,  pour  off  the  aqueous  fluid  and  preserve  for 
further  examination  for  glycerin.  Break  up  the  fatty  acids, 
into  small  pieces  with  a  glass  rod,  place  them  on  the  filter  and 
wash  with  distilled  water  until  the  wash-water  no  longer 
gives  any  reaction  for  sulphuric  acid.^  Then  put  the  fatty 
acids  in  an  evaporating-dish,  place  this  on  the  water-bath^ 
and  heat  till  they  are  melted.  Let  cool  perfectly,  and  free 
the  cake  of  fatty  acids  thus  obtained  from  the  adhering  water 
by  means  of  absorbent  paper.  These  fatty  acids  form  a  mix- 
ture of  oleic  acid,  CigHg^Oa  (fluid  fatty  acid),  palmitic  acid,, 
C16H32O2,  and  stearic  acid,  CisHgeOj  (solid  fatty  acids). 

Reactions  with  Small  Quantities  of  the  Fatty  Acids. 

1.  Action  on  paper,  same  as  in  the  case  of  fat. 

2.  Conduct  towards  the  alkaline  solution  of  rosolic  acid' 
or  phenolphthalein.  The  rosolic  acid  solution  turns  yellow, 
and  the  phenolphthalein  solution  becomes  colorless.  Even 
a  fairly  large  amount  of  tenth-normal  sodium  hydroxide 
solution  may  be  added  without  restoring  the  red  color:  the 
fatty  acids  react  acid. 

3.  Conduct  on  heating  with  monopotassiimi  sulphate:  no 
acrolein  is  formed. 

^  By  20  per  cent,  sulphuric  acid  is  always  meant  one  that  contains  200' 
grams  of  concentrated  sulphuric  acid  in  one  liter. 

*  The  fatty  acids  thus  prepared  are  not  perfectly  pure.  They  always 
contain  some  potassium  sulphate  and  some  soap.  If  it  is  desired  to  have 
the  fatty  acids  free  from  these  substances,  they  must  either  be  repeatedly 
melted  with  water,  or,  more  simply,  extracted  with  ether,  the  ether  ex- 
tract shaken  with  water,  and  the  ethereal  solution  distilled  or  evaporated.. 


EXAMINATION  OF  ADIPOSE  TISSUE.  145 

4.  Conduct  on  heating  with  a  half -saturated  sodium  car- 
bonate solution:  the  fatty  acids  dissolve,  carbon  dioxide  is 
evolved,  and  a  sodium  soap  is  formed.  Cool  the  test-tube  in 
water:  the  solution  soUdifies  to  a  jelly  of  so-called  soap 
gelatin. 

5.  Pour  100  cc.  of  water  upon  2  g.  of  fatty  acids,  heat  and 
dissolve  the  fatty  acids  by  neutrahzing  with  a  solution  of 
sodium  hydroxide:  soap  solution.  The  following  reactions 
are  to  be  made  with  small  portions  of  this  solution  while  it 
is  still  warm. 

(a)  Addition  of  hydrochloric  acid:  precipitation  of  fatty 
acids. 

(6)  Addition  of  calcium  chloride  solution:  insoluble  cal- 
cium soap  is  formed  and  the  solution  loses  the  property  of 
foaming  when  shaken. 

(c)  Addition  of  lead  acetate:  white  precipitate,  which 
becomes  viscous  and  sticky  on  warming;  lead  plaster. 

(d)  Pour  on  a  few  cubic  centimeters  of  the  soap  solution 
some  drops  of  a  vegetable  oil  or  cod-liver  oil  and  shake  once 
or  twice:  homogeneous  milky  fluid  due  to  the  formation 
of  an  emulsion.  The  soaps  possess  in  a  high  degree  the 
property  of  emulsifying  fats.  Repeat  the  last  experiment, 
using,  however,  instead  of  the  soap  solution,  four  drops  of 
sodium  carbonate  solution;  emulsion  often  takes  place  in 
this  case  also,  but  only  when  the  fat  contains  free  fatty  acids, 
for  then  soap  is  formed  from  the  fatty  acids  and  the  sodium 
carbonate.  Absolutely  neutral  fats  containing  no  free  fatty 
acids  are  not  emulsified  when  treated  with  sodium  carbonate 
solution. 

(e)  Place  some  fatty  acids  in  a  dry  test-tube,  and  in 
another  tube  about  the  same  amount  of  fat.  Place  both 
tubes  in  a  beaker  partly  filled  with  water,  heat  the  beaker  on 
the  wire  gauze,  stirring  the  water  constantly  with  a  glass  rod 
(having  a  piece  of  rubber  tubing  on  its  end),  in  order  to 


146  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

obtain  the  most  even  distribution  of  temperature  possible: 
the  fat  melts  sooner  than  the  corresponding  fatty  acid,  i.e., 
the  melting-point  of  the  first  is  lower.  This  is  an  invariable 
rule. 

Separation  of  the  Solid  Fatty  Acids  from  Oleic  Acid. 

Heat  the  remainder  of  the  fatty  acids  in  a  beaker  on  the 
water-bath  till  melted,  then  add  100  cc.  of  70  per  cent,  alcohol, 
continue  heating  somewhat  longer,  filter  while  hot  into  a  dish 
or  beaker,  and  let  cool  completely.  A  paste  of  crystallized 
soUd  fatty  acids  forms,  while  the  oleic  acid,  together  with  a 
part  of  the  solid  fatty  acids,  remain  in  solution.  Dilute  the 
pasty  mass  with  200  cc.  of  70  per  cent,  alcohol,  filter  through 
a  dry  filter,  wash  with  some  70  per  cent,  alcohol,  and  pre- 
serve the  filtrate.  Press  the  solid  fatty  acids  dry  between 
filter-paper.  The  filtrate,  when  evaporated  on  the  water- 
bath,  yields,  when  cold,  a  salve-hke  mass  consisting  of  oleic 
acid  mixed  with  some  sohd  fatty  acids. 

The  preparation  of  pure  oleic  acid  as  well  as  the  separa- 
tion of  palmitic  from  stearic  acid  require  somewhat  more 
detailed  methods  of  procedure. 

Preparation  of  Oleic  Acid. 

The  semi-solid  fatty  acids  are  dissolved  by  heating  with 
sodium  carbonate  solution  and  a  considerable  quantity  of 
water  (clear  solution),  and  neutral  lead  acetate  is  added  to 
the  solution  as  long  as  a  precipitate  forms.  The  mixture  is 
then  faintly  acidified  with  acetic  acid.  The  lead  salts  sepa- 
rate in  viscous  lumpy  masses.  Poiu"  off  the  supernatant 
fluid,  knead  the  lead  salts  with  warm  water,  decant  and 
remove  the  water  adhering  by  heating  on  the  water-bath. 
When  cold  break  up  the  lead  plaster  into  small  pieces,  grind 
it  with  about  three  times  its  volume  of  gypsum  or  caolin 
(slightly  burnt  and  ground  clay)  or  sand,  place  the  mixture 


EXAMINATION  OF  ADIPOSE  TISSUE.  147 

in  a  dry  flask,  pour  on  two  to  three  times  the  volume  of  ether, 
and  let  stand,  shaking  repeatedly,  till  next  day.  Precipitate 
the  lead  completely  from  the  filtered  ethereal  solution  by 
means  of  hydrochloric  acid,  place  the  ethereal  solution  in  a 
separating-funnel,  and  shake  repeatedly  with  water.  The 
solution,  when  separated  and  filtered  through  a  dry  filter, 
fields  oleic  acia  on  distillation  or  evaporation  of  the  ether. 
The  purification  of  the  oleic  acid  depends  upon  the  solubility 
of  the  lead  oleate  in  ether  and  the  insolubiUty  of  the  lead 
palmitate  and  stearate.  Some  of  the  lead  palmitate  and 
stearate  always  dissolves,  however,  and  some  of  the  oleate 
remains  undissolved  in  the  residue. 

Separation  of  Palmitic  from  Stearic  Acid. 
■  Dissolve  the'soUd  fatty  acids  in  95  per  cent,  alcohol  (to 
each  gram  20  cc.  of  alcohol),  then  take  a  tenth  of  the  solution 
and  determine  how  much  of  an  alcoholic  solution  of  neutral 
lead  acetate  is  necessary  for  complete  precipitation.  Measure 
off  nine  times  as  much  of  the  same  lead  acetate  solution  and 
divide  it  into  five  equal  parts.  Add  the  first  fifth  to  the 
alcoholic  solution  of  the  fatty  acids,  filter,  then  precipitate 
with  the  second  fifth,  etc.  (fractional  precipitation).  Each 
precipitate  is  washed  with  cold  alcohol,  pressed  between 
filter-paper,  decomposed  with  hydrochloric  acid,  and  extracted 
with  ether.  The  ether  extract  is  washed  with  water,  the  ether 
evaporated,  and  the  melting-point  *  of  the  residue  determined. 
A  complete  separation  of  palmitic  from  stearic  acid  is  only  to 
be  attained  by  repeating  the  fractional  precipitation  several 
times.  That  the  solid  fatty  acids  form  a  mixture  of  different 
acids  may  also  be  shown  in  a  very  simple  way  by  allowing  a 
hot  solution  of  5  g.  of  the  fatty  acids  in  100  cc.  of  95  per  cent, 
alcohol  to  stand  till  next  day,  filtering  off  the  fatty  acids 

'  Sheepnfat  gives  more  solid  fatty  acids,  especially  stearic  acid,  than 
hog-fat. 


148  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

which  crystallize,  pressing  out  the  mother-Uquor  between 
filter-paper  and,  when  dry,  determining  the  melting-point:  this 
should  be  about  66°.  By  evaporating  the  alcohohc  mother- 
Hquor  an  acid  having  a  melting-point  of  56°  will  be  obtained. 
Both  preparations  are  mixtures  of  palmitic  and  stearic  acids. 
In  the  first  stearic  acid  is  in  excess,  in  the  second  palmitic  acid. 

Separation  of  Glycerin,  C3H5(OH)3. 

The  aqueous  solution  obtained  from  the  soap  solution  by 
precipitating  the  fatty  acids  contains  glycerin,  besides  potas- 
sium sulphate  and  free  sulphuric  acid.  Filter,  nearly  neutral- 
ize with  sodium  hydroxide  solution,  and  then  completely  neu- 
tralize with  sodium  carbonate  solution,  evaporate,  as  nearly  to 
dryness  as  possible,  at  first  over  a  free  flame,  then  on  the  water- 
bath,  and  mix  the  residue  with  50  cc.  of  90  per  cent,  alcohol. 
Filter  the  solution  after  it  has  stood  for  some  time,  evaporate 
again  on  the  water-bath  as  completely  as  possible,  and  dis- 
solve the  residue  in  absolute  alcohol,  so  that  the  volume  of 
the  mixture  amounts  to  25  cc.  in  all.  Without  filtering  add 
25  cc.  of  ether,  shake  thoroughly,  and  let  stand  for  some  time, 
preferably  till  next  day.  The  ether  precipitates  the  greater 
part  of  the  remaining  salts.  Filter  and  evaporate  the  ethereal- 
alcoholic  filtrate  cautiously  by  gently  warming  on  a  water- 
bath.  The  glycerin  is  obtained  in  the  form  of  a  light  yellow- 
colored  sirup  having  an  intensely  sweet  taste. 

Reactions  of  Glycerin. 

1.  Mix  in  a  watch-glass  a  drop  of  glycerin  with  a  little 
borax  and  heat  the  mixture  on  a  platinum  wire  in  the  Bunsen 
flame.  The  flame  is  colored  green  for  a  short  time  (forma- 
tion of  the  glycerin  ester  of  boric  acid).^ 

^  A  better  method  of  performing  this  experiment  is  to  mix  a  little 
borax  and  glycerin  in  a  small  porcelain  dish  or  crucible,  heat  gently  with 


EXAMINATION  OF  ADIPOSE  TISSUE.  149 

2.  Try  the  acrolein  test  with  a  few  drops  of  the  glycerin 
(see  page  142). 

3.  Dilute  the  rest  of  the  glycerin  with  water  (clear  solu- 
tion), add  a  Uttle  sodium  hydroxide  and  then  a  few  drops  of 
copper  sulphate.  The  cupric  hydroxide,  which  first  sepa- 
rates, dissolves,  forming  a  deep-blue  fluid.  This  solution  does 
not  give  any  cuprous  oxide  on  heating,  but  remains  unchanged 
(distinction  from  many  sugars,  especially  glucose). 

a  small  Bunsen  flame  for  a  few  minutes,  add  a  little  alcohol,  and  ignite. 
The  alcohol  bums  \nth  a  yellow  flame  (due  to  the  sodium  in  the  borax) 
in  which  the  green  color  (due  to  the  boric  acid  glycerin  ester)  may  be 
plainly  seen.  The  object  of  heating  the  glycerin  and  borax  previous  to 
the  addition  of  the  alcohol  is  for  the  purpose  of  forming  the  glycerin 
borate. — O. 


CHAPTER  XV. 


5fOLK   AND   WHITE   OF   THE   EGG. 

(a)  Yolk  of  the  Egg. 

I.  Separation  of  the  Yolk  into  its  Constituents. 
11.  Preparation  of  Vitellin;  Detection  of  Lecithin. 

(b)  White  of  the  Egg. 
I.  Reactions  of  Egg-albumin. 

II.  Detection  of  Glucose  in  the  White  of  the  Egg. 
III.  Preparation  of  Ovomucoid. 

(a)   YOLK  OF  THE  EGG. 
I.  Separation  of  the  Yolk  into  its  Constituents. 

Shake  the  yolks  of  several  hen's  eggs  with  ether. 


Ether  extract  A;  reserve  a  small 
quantity,  distil  the  rest;  boil 
the  residue  with  barium  hydrox- 
ide solution. 


Residue  F:    digest  with  gastric 
juice. 


Insoluble  residue    Solution  E :  treat    Solution  G :  albu-    Residue  H:  para- 


B:   treat  with 
ether. 


with  CO2  and 
filter;  contains 
glycerin-phos- 
phoric acid, 
choline  and 
glycerin. 


moses  and  pep- 
tone. 


nuclein  or  para- 
nucleic  acid. 


Ether  extract  C :       Residue  D : 
cholesterin,|lutein.      barium  soaps. 


150 


\.. 


YOLK  AND  WHITE  OF  THE  EGG.  151 

Shake  A'igorously  the  yolks  of  ten  hen's  eggs  in  a  flask  or  in 
a  W'ide-necked  separating-funnel  with  two  to  three  times  their 
vohime  of  ether  and  draw  off  the  yellow-colored  ether  solution 
as  soon  as  it  has  thoroughly  separated.  If  this  separation  does 
not  take  place  readily,  add  a  little  alcohol.  Filter  the  ether 
solution  through  a  dry  filter  and  repeat  the  extraction  with 
fresh  portions  of  ether  until  the  ether  has  only  a  faint  yellow 
color.  The  amouht  of  ether  used  may  be  considerably 
lessened  if  the  successive  ether  extracts  are  distilled  and  the 
distilled  ether  used  over  again.  A  small  portion  of  the  first 
ether  extract  is  reserved  for  the  reactions. 

The  main  quantity  of  the  ether  extract  A  is  distilled,  the 
residue,  while  still  hot,  is  removed  from  the  flask  to  an  evapo- 
rating-dish,  preferably  an  enameled  iron  dish,  the  flask  rinsed 
vnth  small  quantities  of  ether,  and  the  ether  removed  by 
evaporation  on  the  w^ater-bath.  Boil  the  residue  persist- 
ently with  50  g.  of  crystalUzed  barium  hydroxide  and  400  cc. 
of  water,  replacing  from  time  to  time  the  water  which  evapo- 
rates. In  this  process  the  fat  is  split  up  into  fatty  acids, 
which  form  insoluble  barium  soaps,  and  glycerin;  the  lecithin 
into  fatty  acids  (barium  soaps),  glycerin-phosphoric  acid,  and 
choline.  

Tlie  complete  saponification  is  attained  with  difficulty  and  yet  it 
is  very  desirable  for  the  further  work.  It  is  best  to  remove  from  the 
aqueous  fluid  the  sticky  lumps  of  barium  soaps  which  separate  and 
which  contain  much  undecomposed  substance,  extract  them  with  ether, 
evaporate  the  ether  extract,  and  boil  the  residue  again  with  barium 
hydroxide  solution.  The  decomposition  is  complete  when  a  portion 
of  the  ether  extract  of  the  barium  soaps,  after  evaporating  the  ether, 
no  longer  gives  the  acrolein  reaction  (see  chapter  on  Adipose  Tissue, 
page  142). 

Finally  the  barium  soaps  are  removed  from  the  aqueous 
fluid  by  filtration  and  washed. 

The  crude  barium  soap  B  is  freed  from  water  as  far  as 


152  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

possible  by  heating  in  an  evaporating-dish  (and  also  in  part 
by  pcfuring  off  the  water),  then,  when  it  is  perfectly  cold,  it  is 
broken  up  as  finely  as  possible,  placed  in  a  flask,  treated  sev- 
eral times  with  ether  until  it  is  almost  entirely  decolorized, 
and  then  filtered.     X  |^  — — 

The  ether  ex:tr'act  C  is  distilled;  on  standing  the  residue 
yields  cholesterin,  which  is  pressed  between  filter-paper  and 
identified  by  the  chloroform  sulphuric  acid  reaction  (see 
chapter  on  BiUary  Calcuh,  page  91).  The  paper,  which 
was  used  to  free  the  cholesterin  from  its  mother-liquor,  is 
extracted  with  ether,  and  this  is  examined  with  the  spectro- 
scope (lutein).  /When  the  treatment  with  baryta-water  is 
long  continued,  nowever,  the  lutein  is  frequently  so  changed  ( 
that  it  cannot  be  detected  in  this  way  with  certainty.?  It  is 
therefore  advisable  to  use  the  reserved  part  of  the  original 
ether  solution  for  its  detection.  When  this  is  examined 
spectroscopically  the  blue  of  the  spectrum  appears  com- 
pletely absorbed;  on  diluting  with  ether  to  the  proper  point 
an  absorption-band  between  the  green  and  the  blue  appears, 
and  also  a  suggestion  of  a  second  band  in  the  blue/\J^-  por- 
tion of  the  ether  extract  quickly  becomes  colorless  when  nitric 
acid  is  added,  after  giving  a  transient  green  color.  Evapo- 
rate the  remainder  of  the  reserved  part  of  the  ether  solution 
on  the  water-bath  and  dissolve  the  residue  in  a  little  chloro- 
form. 

Shake  a  part  of  the  chloroform  solution  with  a  dilute  solu- 
tion of  sodium  hydroxide:  the  coloring  matter  is  not  with- 
drawn from  the  chloroform  by  the  alkali  (distinction  from 
bilirubin  or  hsematoidin) . 

To  another  part  of  the  chloroform  solution  add  some 
strong  nitric  acid  and  shake:  at  first  a  blue  color  and  then 
decolorization. 

The  residue  D  is  ground  in  a  mortar  with  an  excess  of 
hydrochloric  acid,  the  pasty  mass  placed  in  a  separating- 


YOLK  AND   WHITE  OF   THE  EGG  153 

funnel,  some  more  water  added,  and  the  mixture  extracted 
with  ether.  Separate  the  aqueous  solution  containing 
barium  chloride,  and  wash  the  ether  extract  several  times 
with  water.  The  ether  leaves,  on  evaporating,  fatty  acids 
(in  regard  to  the  methods  of  identifying  these  see  chapter  on 
Adipose  Tissue,  page  144). 

^jrhe-solutTtrrrE  is  freed  from  the  excess  of  barium  hydrox- 
ide by  passing  in  carbon  dioxide  and  filtering  from  the  barium 
carbonate.  The  filtrate  contains  glycerin,  glycerin-phos- 
phoric acid,  and  choHne.  It  is  evaporated  on  the  water-bath 
as  completely  as  possible.  To  show  the  presence  of  the 
glycerin-phosphoric  acid,  C3H5(OH)2P04H2,  grind  a  part  of 
this  residue  with  several  times  its  volume  of  the  oxidizing 
mixture,  heat  in  a  crucible  till  fused,  and  show  the  presence 
of  phosphoric  acid  in  the  fused  mass  by  means  of  ammonium 
molybdate  (see  chapter  on  Milk,  page  9).  Since  barium 
phosphate  is  insoluble  in  water,  the  detection  of  phosphoric 
acid  in  this  case  proves  the  presence  of  an  acid  containing 
phosphorus,  which  forms  a  soluble  barium  salt.  Such  an 
acid  is  glycerin-phosphoric  acid. 


r^       ,  ,11.         HOCH2.CH2I 

To  detect  the  chohne,        .prj  x 


NOH,  extract  the 


greater  part  of  the  residue  resulting  from  the  evaporation 
of  solution  E  with  absolute  alcohol/  precipitate  the  solution 
with  platinum  chloride,  filter  off  the  precipitate,  wash  with 
alcohol,  and  crystalHze  the  choline  platinum  chloride  from 
water.  This  crystallizes  in  large  orange-red  prisms  or  hex- 
agonal plates. 

The  residue  F  is  freed  from  ether  by  grinding  in  a  mortar 

'  Barium  glycerin-phosphate  remains  undissolved,  but  a  considerable 
portion  of  the  glycerin-phosphoric  acid  always  passes  into  solution,  pre- 
sumably as  choline  glycerin-phosphate,  as  the  choline  acts  like  ammonium 
carbonate  in  the  presence  of  carbonic  acid,  i.e.,  it  precipitates  barium 
carbonate  from  the  barium  glycerin-phosphate. 


154  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

and  then  digested  twenty-four  to  forty-eight  hours  with  one 
Hter  of  artificial  gastric  juice  (see  chapter  on  Digestion, 
page  40).  The  albumin  passes  into  solution  as  acid  albu- 
min, albumose,  and  peptone,  and  an  insoluble  residue  of 
paranuclein  or  a  mixture  of  paranuclein  and  paranucleic 
acid  ^  remains.  Filter  this  off,  wash  with  water,  alcohol,  and 
ether,  and  show  the  presence  of  phosphorus  by  fusing  with 
soda  and  saltpeter  (see  chapter  on  Milk,  page  9). 

II.  Preparation  of  Vitellin,  Detection  of  Lecithin  Direct. 

Shake  vigorously  the  yolks  of  two  fresh  eggs  in  a  wide- 
necked  glass-stoppered  vessel  with  200  cc.  of  pure  ether 
which  is  free  from  acid,  and  then  add  5  cc.  of  alcohol.  The 
addition  of  the  alcohol  causes  a  viscous,  slimy  precipitate  ^ 
to  settle  from  the  turbid  mixture.  Pour  off  the  ether  solu- 
tion as  completely  as  possible  and  add  100  cc.  of  a  15  per  cent. 
sQdLmn  chloride  solution  to  the  precipitate.  On  shaking  the 
precipitate  dissolves  in  the  salt  solution,  forming  a  somewhat 
turbid  fluid;  place  the  fluid  in  a  separating-funnel  and  shake 
it  with  an  equal  volume  of  ether.  It  will  then  become  almost 
clear.  Separate  the  aqueous  fluid  and  let  it  stand  till  next 
day;  usually  the  fluid  becomes  turbid  again.  Remove  this 
turbidity  by  shaking  again  with  ether.  Draw  off  the  aqueous 
fluid  again,  measure  it  and  pour  it  into  ten  times  its  volume 
of  water.  Filter  off  the  precipitate,  which  forms,  next  day, 
and  wash  with  water  and  then  with  alcohol.  In  this  con- 
dition the  precipitate  will  contain  a  considerable  quantity  of 

^This  residue  sometimes  contains  a  very  large  amount  of  phosphorus, 
as  much  as  9  per  cent. 

^  Frequently  a  precipitate  forms  even  on  shaking  with  ether  alone ; 
this  precipitate  is  alwaj^s  flocculent  and  is  insoluble  in  sodium  chloride 
solution.  When  this  phenomenon  is  observed  further  work  with  the 
material  is  useless.  Presumably  the  age  of  the  eggs  has  some  influence 
on  their  conduct. 


YOLK  AND  WHITE  OF  THE  EGG.  155 

lecithin,  but  it  is  not  known  whether  this  is  chemically  com- 
bined uith  the  nucleoalbumin,  vitellin,  or  only  adheres  to  it. 
Place  the  precipitate  in  a  flask  and  boil  on  the  water-bath 
with,  absolute  alcohol,  filter,  wash  with  alcohol,  then  with 
ether,  and  finally  grind  in  a  mortar  or  place  in  a  vacuum  desic- 
cator over  sulphuric  acid  to  remove  the  ether.  A  fine  white 
or  Ught-yellow  powder,  which  contains  only  0.95  per  cent,  of 
phosphorus,  is  obtained.  Its  solubility  is  essentially  differ- 
ent from  that  of  the  first  precipitate,  which  still  contains 
lecithin;  presumably  the  vitellin  is  coagulated  by  boiling 
with  alcohol.  No  method  is  yet  known  by  which  the  vitellin 
may  be  freed  from  lecithin  without  coagulating  it.^ 

The  alcoholic  solution  yields  on  evaporation  on  the  water- 
bath  a  yellow  viscous  residue  which  consists  essentially  of 
lecithin, 

(h)  The  White  of  the  Egg. 

The  albimien  or  white  of  the  egg  consists  principally  of  a 
concentrated  solution  (about  11  to  12  per  cent.)  of  a  specific 
proteid,  ovalbumin  or  egg-albumin,  which  is  enclosed  in  a  net- 
work of  membranes  much  less  in  quantity.  Besides  the  egg- 
albumin  there  are  also  present  very  small  quantities  of  a 
globulin,  also  ovomucoid,  glucose,  and  inorganic  salts. 

I.  Reactions  of  Egg-Albumin. 

For  these  reactions  shake  vigorously  20  cc.  of  albumen 
with  150  cc.  of  water  in  a  flask  and  then  filter.  The  solution 
(about  1.5  per  cent.)  must  be  clear  or  only  faintly  opalescent. 
The  first  portions  of  the  filtrate  arc  frequently  turbid;   these 

'  It  might  be  supposed  that  vitellin  could  be  prepared  in  connection 
with  the  separation  of  the  yolk  of  egg  into  its  constituents  given  under  I, 
but  it  has  been  found  that  this  is  generally  not  advantageous,  as  the 
residue  left  when  large  quantities  are  worked  up  does  not  as  a  rule  dis- 
solve easily  in  sodium  chloride  solution;  presumably  this  is  due  to  the 
length  of  the  treatment. 


156  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

are  poured  back  on  the  filter  till  they  run  through  clear.  Test 
the  reaction  and  repeat  the  experiments  described  under 
blood-serum  (see  chapter  on  Blood,  page  60).  The  reac- 
tions of  egg-albumin  are  very  similar  to  those  of  the  dilute 
serum;   they  differ  from  them,  however,  in  some  particulars. 

1.  On  heating  to  boiUng,  the  solution  becomes  milky  and 
the  turbidity  is  more  pronounced  than  in  the  case  of  serum 
solution;  a  separation  of  coagulated  albumin,  however,  does 
not  take  place.  The  cautious  addition  of  acetic  acid  brings 
about  the  coagulation.  The  precipitate  is  not  so  flocculent 
as  in  the  case  of  serum  albumin,  but  appears  somewhat 
swollen.  A  further  addition  of  acetic  acid  dissolves  the  pre- 
cipitate, but  not  so  readily  as  in  the  case  of  serum  albumin.^ 
Repeat  experiments  2,  3,  and  4,  under  Blood-serum,  with  the 
egg-albumin  solution. 

5.  Heat  a  portion  of  the  solution  with  half  its  volume  of 
sodium  hydroxide  solution:  alkali  albuminate  is  formed. 
Neutralize  the  cooled  solution  with  dilute  sulphuric  or  acetic 
acid:  the  albuminate  precipitates.  In  excess  of  the  acid 
this  dissolves  far  more  difficultly  than  in  the  case  of  serum 
albumin. 

6  and  7  as  with  the  serum. 

8.  If  a  portion  of  the  solution  is  treated  with  nitric  acid 
till  a  permanent  precipitate  is  formed  and  then  absolute 
alcohol  is  added  till  the  volume  has  been  doubled,  the  pre- 
cipitated albumin  does  not  dissolve,  or  dissolves  only  very 
slightly  (distinction  from  serum  albumin). 

9.  If  strong  nitric  acid  of  the  specific  gravity  1.48  be 
added  to  a  little  of  the  solution,  a  precipitate  is  formed  which 
does  not  dissolve  when  the  quantity  of  the  nitric  acid  added 
amounts  to  about  half  of  the  volume  of  the  albumin  solution 


^  The  expression  serum  albumin  is  here  used  only  for  the  sake  of  brev- 
ity in  place  of  "proteids  of  the  blood-serum" 


YOLK  AND  WHITE  OF  THE  EGG.  157 

used.  In  order  to  dissolve  the  precipitate  a  much  greater 
addition  of  acid  or  heating  of  the  solution  is  necessary  (dis- 
tinction from  serum  albumin). 

10.  On  shaking  a  small  quantity  of  the  solution  with  an 
equal  volume  of  ether,  coagulation  gradually  takes  place. 

11.  On  heating  a  small  portion  of  the  solution  after  the 
addition  of  an  equal  volume  of  sodium  hydroxide  of  1.34 
specific  gravity  and  about  three  drops  of  neutral  lead  acetate, 
it  turns  black.  The  blackening  is  more  pronounced  than  in 
the  case  of  serum  albumin.  If  this  solution  is  then  acidified 
with  hydrochloric  acid  there  results,  not  a  turbid  grayish- 
yellow  fluid  as  in  the  case  of  serum  albumin,  but  coarse  dark- 
gray  flakes  precipitate,  while  the  fluid  becomes  almost  clear. 
This  distinction  is  due  to  the  fact,  first,  that  more  sulphur  is 
spUt  off  from  the  egg-albumin,  and,  secondly,  that  the  albu- 
minate from  the  egg-albumin  dissolves  with  more  difficulty 
in  hydrochloric  acid  than  the  albuminate  from  serum  albumin. 

The  reactions  of  a  solution  ten  times  as  dilute  agree 
entirely  with  those  given  for  the  correspondingly  dilute 
serum. 

II.  Detection  of  Glucose. 

Shake  up  the  white  of  a  hen's  egg  with  ten  times  its 
volume  of  water  (about  200  cc),  add  acetic  acid  to  neutral 
reaction,  and  heat  to  vigorous  boiling  in  a  large  evaporating- 
dish  over  a  free  flame  and  with  constant  stirring  (caution  on 
account  of  the  strong  foaming)  until  the  albumin  has  sepa- 
rated in  lumps  and  the  fluid  appears  quite  clear.  Then  filter, 
wash  with  some  water,  and  evaporate  the  filtrate  and  the 
wash-water  over  a  free  flame  to  a  small  volume,  about  10  to  12 
cc.  Use  half  of  this  for  the  Trommer's  test  with  sodium 
hydroxide  and  copper  sulphate  solutions,  and  the  other  half 
for  the  fermentation  test.     Both  give  positive  results. 


158  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

III.  Preparation  of  Ovomucoid. 

There  is  present  in  the  albumen  of  the  hen's  egg,  besides 
the  ovalbumin,  a  mucin-like  substance,  in  considerable 
quantity  (about  one-eighth  of  the  organic  part  of  the  dry- 
substance).  This  does  not  coagulate  and  is  characterized 
by  its  very  peculiar  physical  conduct. 

In  order  to  prepare  it,  add  to  the  albumen  of  three  hen's 
eggs  four  times  the  volume  of  water,  shake  thoroughly,  filter, 
pour  the  filtrate  into  1.5  times  the  volume  of  boiling  water, 
add  acetic  acid  to  neutral  or  very  slightly  acid  reaction, 
and  heat  with  constant  stirring  over  a  free  flame  to  vigorous 
boiling.  Filter,  evaporate  the  filtrate  (which  should  give 
no  precipitate  with  mercuric  chloride  solution)  at  first  over 
a  free  flame  and  then  on  the  water-bath  to  about  20  cc' 
Filter  the  solution  again,  if  necessary,  and  pour  it  into  100  cc. 
of  absolute  alcohol,  filter,  wash  the  precipitate  once  with 
ordinary  alcohol,  then  once  with  absolute  alcohol,  finally  with 
ether,  and  dry  by  allowing  the  ether  to  evaporate  in  the  air. 

The  ovomucoid  thus  obtained,  which  forms  a  fine  white 
powder,  is  dissolved  in  100  cc.  of  water  and  the  solution 
divided  into  three  equal  parts. 

1.  Evaporate  one  part  of  the  solution  to  dryness  on  the 
water-bath:  a  substance  resembling  horn  is  left.  When 
this  is  covered  with  water  and  allowed  to  stand  it  swells  and 
forms  a  jelly-like  mass. 

2.  Mercuric  chloride  solution  gives  no  precipitate;  but  a 
solution  of  tannin  and  also  phosphotungstic  acid  with  hydro- 
chloric acid  give  precipitates.   Millon's  reaction  is  also  positive. 

3.  To  the  third  part  of  the  solution  add  7  to  8  cc.  of 
hydrochloric  acid,  heat  to  boiling,  keep  boiling;  gently  for  five 
minutes,  let  cool,  neutralize  and  make  the  test  for  sugar 
according  to  Trommer  and  also  with  the  freshly  mixed 
Fehling's  solution.  The  separation  of  the  cuprous  oxide  as 
a  rule  takes  place  only  after  cooling  or  allowing  to  stand. 


CHAPTER  XVI. 

EXAI^IINATION  OF  THE  PRODUCTS  OF  THE  PUTREFACTION 

OF  PROTEIDS. 

I.  Abridged  Method. 

Five  hundred  grams  of  chopped  meat,  2  hters  of  water,  and 
60  cc.  of  a  cold  saturated  solution  of  sodium  carbonate  are 
placed  in  a  bottle,  thoroughly  mixed  by  shaking,  and  digested 
at  40°  for  6  to  8  days;  the  bottle  being  loosely  closed  by 
means  of  a  plug  of  cotton.  When  the  time  given  has  elapsed, 
subject  the  entire  mass  to  distillation  without  the  addition 
of  acid.  When  the  contents  of  the  distilling-flask  or  retort 
become  somewhat  thick,  let  cool,  add  another  liter  of  water, 
and  distil  again.  The  distillate  and  residue  are  worked  up 
separately. 

(a)  Treatment  of  the  Distillate.* 
The  distillate  is  acidified  with  hydrochloric  acid  and  extracted  with  ether. 


Ether    solution    (A)    shaken    with         Aqueous  fluid  (B)  evaporated, 
sodium  hydroxide  solution. 


I  I 

Ether  solution  (C)  evaporated:  Alkaline    solution    (D)    acidified, 

indol  and  also  skatol.  sodium  carbonate  solution  added, 

then  extracted  with  ether. 


Ether     solution     (E)     evaporated:         Alkaline    fluid  (F)    contains    the 
phenol  and  cresol.  sodium  salts  of  the  volatile  fatty- 

acids     (and     some     phenylpro- 
pionic  acid). 


*  Zeitschr.  f .  physiol.  Chemie,  9,  492. 

159 


160  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

The  exti  action  of  the  first  distillate  is  best  doneinasepa- 
rating-funnel  and  in  separate  portions  of  about  300  cc.  with 
200  cc.  of  ether.  After  shaking  vigorously  draw  off  the 
aqueous  fluid  (B)  and  pour  into  the  separating-funnel  a  new 
quantity  of  the  distillate,  etc.  Since  the  ether  is  dissolved 
to  some  extent  by  water,  some  fresh  ether  is  to  be  added  each 
time. 

When  the  entire  distillate  has  been  extracted,  place  all 
the  aqueous  fluid  (B)  in  a  large  dish  and  let  it  stand  until  the 
dissolved  ether  has  volatilized  spontaneously,  then  evaporate : 
there  remains  a  white  mass  of  salts,  which  consist  very  largely 
of  ammonium  chloride. 

The  ether  solution  (A)  is  now  very  thoroughly  shaken 
with  the  same  volume  of  water  and  50  cc.  of  sodium  hydrox- 
ide solution.  The  volatile  acids  formed  in  the  putrefaction 
are  dissolved  in  the  alkaline  solution  (D),  while  indol  and 
also  skatol  remain  in  the  ether  solution.  Distil  off  most  of 
the  ether  from  the  ether  extract  (now  designated  (C)  )  at  a 
gentle  heat  on  the  water-bath,  and  let  the  residue  evaporate 
spontaneously.  Impure  indol  remains.  For  the  reactions 
of  this  substance  see  page  169. 

The  alkaline  solution  (D)  is  again  acidified  with  hydro- 
chloric acid,  sodium  carbonate  solution  added  until  the  fluid 
reacts  acid  only  from  the  carbonic  acid  or  is  neutral  (a  por- 
tion taken  out  and  heated  must  show  an  alkaline  reaction 
when  cold),  and  the  mixture  shaken  with  ether.  Phenol 
and  cresol  are  dissolved  by  the  ether,  while  the  volatile  fatty 
acids  remain  in  the  aqueous  fluid  as  alkali  salts.  As  carbon 
dioxide  is  evolved  on  shaking,  considerable  pressure  develops 
in  the  separating-funnel;  it  is  therefore  necessary  to  be  cau- 
tious and  to  remove  the  stopper  repeatedly  or  to  reverse  the 
separating-funnel  and  open  the  stop-cock.  The  ether  extract 
(E)  is  then  separated  from  the  aqueous  fluid  (F).  The 
ethereal  solution  (E),  when  evaporated,  leaves  an  impure 


PUTREFACTION  PRODUCTS   OF  PROTEIDS.  161 

mixture  of  phenol  and  cresol,  principally  paracresol.  In 
order  to  show  the  presence  of  these  substances,  heat  the  oil 
in  a  flask  with  some  water  and  let  cool. 

1.  Add  some  ferric  chloride  to  a  portion  of  the  solution: 
dirty  bluish-gray  color. 

2.  Warm  a  second  portion  of  the  solution  with  some 
Millon's  reagent :  red  color. 

3.  Add  to  a  third  portion  some  bromine- water ;  precipitate 
of  tribromphenol  and  tribromcresol  (or  other  bromine  com- 
poimds) . 

For  more  detailed  information  concerning  phenol  and 
cresol  see  pages  106  and  171. 

The  aqueous  fluid  (F)  is  again  placed  in  the  separating- 
funnel,  strongly  acidified  with  hydrochloric  acid,  and  extracted 
with  a  small  quantity  of  ether  (caution  on  account  of  the  car- 
bon dioxide  set  free).  The  ethereal  solution  when  drawn  off 
and  evaporated  leaves  the  volatile  fatty  acids,  mixed  with  a 
small  quantity  of  the  homologues  of  benzoic  acid. 

(b)  Treatment  of  the  Distillation  Residue. 
The  distillation  residue  is  evaporated,  precipitated  -nith  alcohol,  and  filtered. 


Filtrate    (A)    evaporated,    treated         Residue  (B) :  undissolved  albumin, 
A\ith  sulphuric  acid  and  ether.  bacteria,    albumoses,     peptone, 

I  and  salts. 


Ether    extract    (C)    distilled    and         Aqueous  solution  (D)  contains  al- 
treated   A\-ith   sodium  hydroxide  bumoses  and  peptone, 

and  barium  chloride  solutions. 


Precipitate  (E) :   barium  soaps.  Filtrate  (F)  evaporated  and  treated 

with  HCl  and  ether. 


Ether  solution  (G)  ev'aporated,the  resi-    Aqueous  solution  (H)  contins  sodi- 
due  boiled  with  water,  and  filtered.         um  chloride  and  other  chlorides. 


Solution   (I)    contains    skatol-car-         Insoluble  oil  (K),  ground  with  zinc 
bonic    acid,    aromatic  oxyacids,  oxide,  then  boiled  and  filtered: 

and  succinic  acid.  zinc  .salts  of  phenyl-propionic  and 

phenyl-acetic  acids  crystallize. 


162  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

The  distillation  residue  has  an  acid  reaction.  In  order  to 
avoid  the  escape  of  phenyl-propionic  and  phenyl-acetic  acid 
with  the  steam  on  evaporating,  it  must  be  made  alkaline 
with  sodium  carbonate  solution.  Since  the  fluid  still  con- 
tains ammonium  salts,  it  must  be  again  made  alkaline  from 
time  to  time.  Evaporate  to  a  sirup,  precipitate  with  several 
times  its  volume  of  alcohol,  and  filter,  best  not  till  next  day, 
from  the  precipitate  (B)  consisting  of  undissolved  albumin,  etc. 

The  filtrate  (A)  is  freed  from  alcohol  by  evaporating  on 
the  water-bath,  the  residue  dissolved  in  150  cc.  of  dilute  sul- 
phuric acid  (20  per  cent,  i.e.,  200  g.  H2SO4  in  the  liter),  and 
repeatedly,  but  not  too  violently,  shaken  with  ether.  The 
ether  frequently  settles  very  slowly,  and  it  is  often  necessary 
to  add  alcohol  to  facilitate  the  separation  of  the  ether. 

The  aqueous  solution  (D)  remaining  contains,  besides 
free  sulphuric  acid,  albumoses  and  peptone. 

The  ether  extract  (C)  is  distilled,  the  residue  dissolved  in 
water  and  sodium  hydroxide  solution  (added  to  alkaline  reac- 
tion), and,  in  order  to  precipitate  the  palmitic,  stearic,  and 
oleic  acids,  barium  chloride  solution  is  added  as  long  as  a 
precipitate  forms,  and  after  allowing  to  stand  awhile  the 
liquid  is  filtered. 

The  precipitate  (E)  consists  of  barium  soaps. 

The  filtrate  (F)  is  evaporated  to  about  100  cc,  placed  in 
a  separating-funnel,  100  cc.  of  hydrochloric  acid  added,  and 
extracted  with  ether. 

The  ether  solution  (G)  is  drawn  off,  distilled,  and  then 
evaporated  on  the  water-bath.  The  oil  remaining  is  washed 
into  a  flask  with  hot  water,  boiled  with  about  100  cc.  of  water, 
cooled  and  filtered. 

The  aqueous  solution  (H)  contains  sodium  chloride  and 
hydrochlorides  of  some  organic  bases.  To  isolate  these,  evapo- 
rate the  solution  as  completely  as  possible  and  extract  the 
residue  with  absolute  alcohol.  After  allowing  to  stand  for 
some  time,  filter  the  solution  and  evaporate  to  dryness  on 


PUTREFACTION  PRODUCTS  OF  PROTEIDS.  163 

the  water-bath.  Extract  the  residue  again  with  absolute 
alcohol  and  continue  this  treatment  until  the  residue  dissolves 
completely  in  absolute  alcohol,  formuig  a  clear  solution.  The 
residue  of  hydrochlorides — principally  of  (5-aminovaleric  acid/ 
C5HJ1XO2HCI, — which  remains  on  evaporation,  solidifies 
gradually  when  meat  has  been  used,  but  when  fibrin  and 
gelatin  have  been  iised  a  crystalhne  mass  results  immediately 
on  cooling. 

The  aqueous  solution  (I)  contains  skatol-carbonic  acid 
and  oxyacids,  as  may  be  proved  by  their  reactions;  the 
skatol-carbonic  acid  by  its  reaction  with  ferric  chloride,  the 
oxyacids  by  their  conduct  with  Millon's  reagent  and  with 
bromine- water  (see  further  page  172). 

The  oil  (K)  insoluble  in  water  is  ground  in  a  mortar  with 
zinc  oxide,  the  mixture  washed  into  a  flask  with  water  and 
heated  to  boiling.  Filter  while  hot.  From  the  filtrate  a 
zinc  salt  very  soon  crystallizes;  usually  this  is  a  mixture  of 
the  zinc  salts  of  phenyl-propionic  and  phenyl-acetic  acids. 

II.  MORE  Detailed  Examination. 

Place  two  kilos  of  blood-fibrin  in  a  large  flask  with  eight 
liters  of  water,  add  2  g.  of  potassium  phosphate  (KH2PO4), 
1  g.  of  crystallized  magnesium  sulphate,  200-240  cc.  of  a 
cold  saturated  solution  of  sodium  carbonate,  and  then  add 
some  macerated  meat  to  start  the  putrefaction.  This  macer- 
ated meat  is  obtained  by  allowing  a  mixture  of  10  g.  of  finely 
chopped  meat,  100  cc.  of  water,  and  1-2  cc.  of  sodium  car- 
bonate solution  to  stand  for  twenty-four  hours  at  40-42°.  Add 
a  few  cubic  centimeters  of  this  mixture  as  well  as  some  of  the 
solid  particles  to  the  contents  of  the  flask.  The  flask  is  then 
closed  with  a  cork  carrying  a  glass  tube  which  is  connected, 
by  means  of  a  rubber  tube,  with  a  wash-bottle  containing  a 
3  per  cent,  solution  of  mercuric  cyanide.  By  this  means 
'  See  H.  Salkowski,  Ber.  d.  d.  chem.  Ges.  31,  776. 


164  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

the  methyl  mercaptan,  discovered  by  Nencki  among  the  gases 
formed  during  putrefaction,  is  collected  in  the  form  of  the 
mercury  mercaptide.  This  arrangement  also  helps  materi- 
ally to  diminish  the  odor  of  putrefaction  in  the  room  in  which 
the  experiment  takes  place. 

Digest  about  six  days  or  even  longer  and  then  distil  the 
mixture,  best  from  a  large  metal  vessel.  Measure  the  dis- 
tillate. As  soon  as  seven  liters  have  distilled  over  pour 
about  2.5  liters  of  water  into  the  distillation  vessel  and 
distil  off  the  same  amount.  The  indol,  skatol,  and  phenol 
pass  over  almost  completely  with  the  strongly  ammoniacal 
distillate.  The  distillate  also  contains — besides  hydrogen 
sulphide  or  ammonium  sulphide,  ammonium  carbonate  and 
ammonium  bases — small  quantities  of  volatile  fatty  and 
aromatic  acids,  while  the  greater  part  of  these  acids  remains 
in  the  distillation  residue  as  the  sodium  salts.  The  method 
of  treatment  of  the  distillate  and  of  the  distillation  residue, 
apart  from  some  slight  modifications,  is  the  same  as  given 
above,  except  that  the  isolation  or  purification  of  the  products 
can  be  carried  further  on  account  of  the  larger  quantities  of 
the  substances  at  command. 

(a)  Distillate. 

1.  To  get  rid  of  the  disagreeable  hydrogen  sulphide  it  is 
advisable  to  add  some  copper  sulphate  solution  to  the  dis- 
tillate and  to  filter  from  the  copper  sulphide.  The  aqueous 
fluid  (B)  (see  scheme  on  page  159)  is  evaporated  as  before, 
but,  since  it  contains  copper  sulphate,  it  is  advantageous  tO; 
distil  the  residue  with  sodium  hydroxide  solution,  collecting 
the  ammonia  and  the  other  bases  in  hydrochloric  acid,  and 
then  evaporate  the  solution  thus  obtained.  If  no  copper 
sulphate  has  been  used,  this  roundabout  way  may  be  dis- 
pensed with.  To  isolate  the  ammonium  bases,  the  residue 
resulting  is  extracted  in  the  usual  manner  with  absolute  alec- 


PUTREFACTION  PRODUCTS  OF  PROTEIDS.  165 

hol,  which  leaves  the  ammonium  chloride  undissolved,  and  the 
ammonium  bases  are  precipitated  with  platinum  chloride,  etc. 

2.  The  indol  obtained  by  evaporating  the  ether  solution 
(C)  is  still  impure,  containing  especially  phenol  or  cresol.  In 
order  to  remove  these  impurities,  wash  the  mixture  into  a 
flask  wth  hot  water,  add  sodium  hydroxide  solution,  and  dis- 
til, preferably  in  a  current  of  steam.  The  indol  passes  over 
into  the  receiver,  partly  as  a  half-melted  white  mass,  partly 
in  the  form  of  leaflets  and  some  deposits  as  a  solid  in  the 
condenser-tube.  To  remove  the  indol  from  this  to  the  receiver 
after  all  the  indol  has  distilled,  it  is  best  to  attach  a  flask  con- 
taining ether  to  the  condenser  in  place  of  the  distilling-fiask 
and  gently  warm  it  on  the  water-bath.  The  ether  which 
,  condenses  in  the  condenser-tube  dissolves  the  indol,  and  the 
ethereal  solution  runs  into  the  receiver;  finally,  the  whole 
of  the  indol  is  extracted  from  the  distillate  with  ether,  the 
ether  solution  separated,  and,  after  distilling  off  most  of  the 
ether,  allowed  to  evaporate  spontaneously.  The  alkaline 
fluid  remaining  in  the  distilling-flask  is  added  to  the  alkaline 
solution  (D). 

Very  frequently  the  indol  thus  obtained  contains  skatol. 
To  detect  this  substance,  it  is  sufficient,  when  it  is  present  in 
any  considerable  quantity,  to  distil  a  portion  of  the  indol 
wth  water:  the  first  drops  of  the  distillate  will  contain  prin- 
cipally skatol  in  the  form  of  leaflets  with  a  mother-of-pearl 
lustre,  as  the  skatol  is  much  more  readily  volatile  with  steam 
than  indol. ^ 

3.  The  crude  mixture  of  phenol  and  cresol  obtained  by 
evaporating  the  ether  solution  (E)  is  also  to  be  purified  by 
distillation  in  steam  after  it  has  been  made  alkaline  with 
sodium  carbonate  solution.  A  small  portion  of  these  sub- 
stances is  lost  in  this  process.  From  the  distillate  the  sub- 
stances in  question  are  again  extracted  with  ether. 

1  Zeitschr.  f .  phys.  Chemie,  8,  438. 


166  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

4.  The  acids  obtained  from  the  alkaUne  fluid  (F)  are  to 
be  combined  with  the  volatile  acids  obtained  from  the  dis- 
tillation residue  (see  further  below). 

(6)  Distillation  Residue. 

On  account  of  the  larger  quantity  of  the  albuminous 
material  used,  the  quantity  of  the  reagents  is  also  to  be  cor- 
respondingly increased  (about  four  times).  The  more 
detailed  treatment  refers  especially  to  the  acids  obtained 
from  the  distillation  residue.  Some  other  modifications 
which  facilitate  the  purification  of  the  individual  products 
of  putrefaction  are,  however,  also  advantageous. 

The  following  scheme  is  a  good  one  to  use:  * 

Treatment  of  the  Distillation  Residue. 
The  residue  is  made  alkaline  with  sodium  carbonate,  evaporated,  and  pre- 
cipitated with  alcohol. 


Precipitate  (A)  (undissolved  albu-        Alcoholic    solution      evaporated, 
min,  bacteria,  and  salts).  acidified  with  sulphuric  acid,  and 

extracted  with  ether. 


Ether  extract  distilled,  made  alka-        Aqueous    solution     (B)     contains 
line  with  NaOH,^  extracted  with  H2SO4,  peptone,  and  basic  sub- 

ether,  stances. 


Ether  extract  (C).  Alkaline  solution  precipitated  with 

BaCL  and  filtered. 


Filtrate  treated  with  HCl  and  ether.         Precipitate  (D)  barixun  soaps. 


Acid  aqueous  solution  (E)  basic  sub-        Ether  extract  (F^  evaporated  and 
stances'.  distilled  with  steam. 


Volatile;    fatty  acids,  homologues  Non- volatile :      oxyacids,     skatol- 
of  benzoic  acid;  collected  in  so-  carbonic  acid,  succinic  acid, 

dium    hydroxide    solution,    HCl 
added,  and  extracted  with  ether. 

^  The  examination  for  ptomaines  is  not  taken  up  in  the  following  scheme. 
In  this  connection  the  reader  is  referred  to  "  Brieger ;  Investigations  on  the 
Ptomaines,"  Berlin,  1885-1886. 

^  The  required  quantity  of  sodium  hydroxide  solution  is  measured  off. 


PUTREFACTION  PRODUCTS  OF  PROTEIDS.  167 

REAf.uiKS. — The  oily  residue  remaining  after  distilling 
the  ether  extract  (F),  which  contains  volatile  acids,  oxyacids, 
skatol-carbonic  acid,  and  succinic  acid,  is  placed  in  a  flask 
and  distilled  in  a  rapid  current  of  superheated  steam.  The 
steam  is  superheated  by  passing  it  through  a  gently  heated 
copper  spiral.*  This  should  not  be  heated  too  strongly,  as 
the  skatol-carbonic  acid  will  then  be  resinified  to  a  considera- 
ble extent.  This  resinification  cannot  be  entirely  avoided, 
however,  in  any  case.  The  vapors  are  conducted  directly 
into  sodium  hydroxide  solution,  which,  of  course,  becomes 
quite  hot. 

It  is  well  not  to  have  the  current  of  steam  too  strong  at 
first,  as  then  too  much  of  the  acid  escapes  unabsorbed.  The 
previous  treatment  of  the  distillation  residue  gives  sufficient 
data  to  estimate  the  quantity  of  sodium  hydroxide  solution 
to  be  placed  in  the  receiver;  use  about  the  quantity  which 
was  needed  to  make  the  first  acid  ether  extract  alkaline.  To 
drive  over  the  volatile  acids  completely  requires  considerable 
time,  twenty-four  to  thirty-six  hours.  As  a  criterion  we 
make  use  of  the  conduct  of  a  very  weak  alkaline  fluid  (con- 
taining 1  to  2  cc.  one-tenth  normal  sodium  hydroxide), 
placed  in  a  receiver:  if  this  fluid  still  remains  alkaline  after 
an  hour,  the  distillation  is  to  be  regarded  as  completed. 

The  entire  alkaline  solution  is  evaporated  on  the  water- 
bath  and,  after  it  is  cold,  strongly  acidified  ^  with  hydro- 
chloric acid  and  extracted  with  ether.  The  residue  left  after 
distilling  the  ether  extract  is  distilled  from  a  flask  provided 
with  a  thermometer.  The  volatile  fatty  acids  distil  first. 
The  receiver  is  changed  when  the  temperature  has  risen  to 
about  260°  and  the  distillation  is  continued  until  only  a  very 

'  Zeitschr.  f.  physiol.  Chemie.  9.  493 

^  To  detect  the  presence  of  free  hydrochloric  acid  test  the  reaction  of 
a  few  drops  of  the  fluid,  after  the  first  extraction  with  ether,  with  methyl 
violet. 


168  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

slight  residue  remains  in  the  flask.  A  mixture  of  phenyl- 
acetic  acid  and  phenyl-propionic  acid  is  thus  obtained,  from 
which  frequently,  but  not  always,  one  of  the  two  acids 
separates. 

A  quantitative  method  for  the  separation  of  these  two 
acids  is  not  known  at  the  present  time.  To  detect  the  two 
we  may  either  make  use  of  their  conduct  in  the  animal  body — 
phenyl-propionic  acid, is  converted  into  hippuric  acid,  phenyl- 
acetic  acid  into  phenaceturic  acid,  which  are  readily  sepa- 
rated ^ — or  of  one  of  the  other  methods  given.  ^  If  it  is  only 
desired  to  prove  the  presence  of  the  homologues  of  benzoic 
acid,  Liicke's  reaction  is  sufficient  (see  page  106). 

The  solution  left  in  the  distilling-flask,  after  driving  out 
the  volatile  acids,  which  contains  skatol-carbonic  acid,  oxy- 
acids,  and  succinic  acid,  gradually  becomes  turbid  on  cooling 
and  deposits  some  resinous  substance.  It  must  be  filtered 
as  soon  as  this  material  has  settled  so  that  filtration  is  possi- 
ble (after  some  hours).  From  the  clear  filtrate  there  deposits 
after  twenty-four  hours'  standing  in  the  cold,  best  in  the 
ice-chest,  chalky-white  grains  of  pure  skatol-carbonic  acid. 
By  evaporating  the  aqueous  solution,  separated  from  the 
skatol-carbonic  acid,  to  half  of  its  volume,  a  new  deposit  of 
skatol-carbonic  acid  is  frequently  obtained;  the  separation 
is,  however,  never  complete,  a  portion  of  the  skatol-carbonic 
acid  always  remaining  in  the  aqueous  solution,  together  with 
the  oxyacids  and  succinic  acid.  The  complete  separation  of 
the  oxyacids  from  succinic  acid  has  also  not  yet  been  accom- 
plished. If  the  aqueous  solution  be  shaken  with  pure  ether, 
the  oxyacids,  together  with  the  skatol-carbonic  acid  still 
present,  are  extracted,  but  some  of  the  succinic  acid  is  also 
taken  up  by  the  ether,  while  the  greater  part  remains  in  the 


*  Zeitschr.  f.  physiol.  Chemie,  9,  503. 

=>  Ibid.  10,  150,  and  H.  Salkowski,  Ber.  d.  d.  chem.  Ges.,  18,  323. 


PUTREFACTION  PRODUCTS  OF  PROTEIDS.  169 

aqueous  solution.  The  aromatic  oxyacids  may  be  obtained 
by  crj'stallizing  from  hot  water  the  residue  remaining  after 
evaporating  the  ether  solution. 

To  separate  the  two  acids,  the  hydroparacumaric  acid 
and  the  paraoxyphenyl-acetic  acid,  their  conduct  towards 
benzene  may  be  utilized  accordmg  to  E.  Baumann.  Both 
acids  are  difficultly  soluble  in  benzene;  the  hydroparacumaric 
acid  is,  however,  more  readily  soluble  than  the  paraoxyphenyl- 
acetic  acid.  A  quantitative  method  of  separation  is  not  yet 
kno'^Ti. 

Properties  and  Reactions  of  the  Products  Obtained. 

I.  Indol,  CeH,<(^^>CH. 

Indol  crystallizes  from  hot  water  in  shining  white  leaf- 
lets, and  is  easily  volatile  with  steam.  Melting-point  52°. 
It  is  difficultly  soluble  in  water,  readily  soluble  in  ether,  alco- 
hol, benzene,  and  chloroform.  If  a  solution  of  picric  acid  in 
benzene  be  added  to  a  solution  of  indol  in  petroleum  ether, 
shining  red  needles  of  a  compound  of  equal  molecules  of  indol 
and  picric  acid  precipitate.  These  when  distilled  with 
ammonia  yield  indol.— Indol,  when  introduced  mto  the 
animal  body,  is  oxidized  to  mdoxyl  and  appears  in  the  urine 

as  potassium-indoxyl  sulphate  (indican),  02S<^9S?"^"-^. 

REACTIONS   OF   INDOL. 

(a)  If  a  cold  saturated  aqueous  solution  of  indol  be  acidi- 
fied with  nitric  acid  and  then  a  few  drops  of  potassium  nitrite 
be  added,  a  flocculent  bright  brick-red  precipitate  of  nitroso- 
indol  nitrate,  according  to  Nencki,  CieHi3(NO)N2.HN03,  is 
formed.  Very  dilute  solutions  of  indol  only  turn  red;  if 
shaken  with  chloroform,  a  red-colored  crust  separates  at  the 
surface  of  contact  of  the  chloroform  with  the  aqueous  fluid. 


170  PHYSIOLOGICAL  AND  .  PATHOLOGICAL  CHEMISTRY. 

(6)  Legal's  Reaction.  Add  a  few  drops  of  sodium  nitro- 
prusside  solution  (freshly  prepared)  till  a  distinct  yellow  color 
appears,  and  then  some  drops  of  sodium  hydroxide  solution : 
deep  violet  color.  On  acidifying  with  acetic  acid  the  fluid 
turns  azure-blue. 

(c)  So-called  "  Cholera-red  Reaction."  Very  dilute  indol 
solutions,  which  also  contain  a  nitrite,  give  a  splendid 
purple  color  with  concentrated  sulphuric  acid.  Cultures  of 
the  cholera  bacilli  also  give  this  color,  as  they  contain  both 
indol  and  a  nitrite.  The  nature  of  the  coloring  matter  is  not 
known.  To  perform  the  reaction  ^  add  to  10  cc.  of  a  very 
dilute  indol  solution  (containing  0.03  to  0  05  part  per  thou- 
sand) 1  cc.  of  a  0.02  per  cent,  potassium  nitrite  solution, 
mix  thoroughly,  and  pour  in  some  concentrated  sulphuric 
acid,  so  that  it  forms  a  layer  on  the  bottom  of  the  test-tube : 
purple  color.  On  neutralizing  with  sodium  hydroxide  solu- 
tion the  fluid  turns  blue-green.  The  reaction  also  takes 
place  on  mixing  with  dilute  sulphuric  acid.  When  used 
with  cultures  it  is  only  to  be  regarded  as  proving  the  pres- 
ence of  cholera  bacilli  and  some  other  kinds  of  bacilli,  when 
the  sulphuric  acid  used  is  absolutely  free  from  nitrous  acid. 

CH, 

2.  Skatol,  Methyl  Indol,  CaB.i<^      "^^CH. 

Colorless,  shining  leaflets,  more  readily  volatile  with  steam 
than  indol,  of  a  penetrating  fecal  odor,  which  is  scarcely  percep- 
tible when  the  skatol  is  pure.  Melting-point  95°.  More  diffi- 
cultly soluble  in  water  than  indol,  readily  soluble  in  ether, 
alcohol,  chloroform,  and  benzene.  When  introduced  into  the 
organism  it  is  oxidized  to  skatoxyl,  which  appears  in  the  urine 

as  potassium  skatoxyl  sulphate,  ^2^\r\Tr    *   ,  (Brieger), 

^E.  Salkowski;   Virchow's  Archiv,  110,  366  (1887). 


PUTREFACTION  PRODUCTS  OF  PROTEIDS.  171 

REACTIONS   OF   SKATOL. 

(a)  Skatol  dissolves  in  concentrated  hydrochloric  acid 
with  a  violet  color. 

(6)  If  the  aqueous  solution  be  acidified  with  nitric  acid 
and  then  a  few  drops  of  potassium  nitrite  solution  be  added, 
no  red  color  results,  as  in  the  case  of  indol,  but  only  a  white 
turbidity. 

3.  Phenol. 

For  the  properties  and  reactions  of  this  substance  see 
chapter  on  Urine,  page  106. 

4.  Paracresol,CeH/^g3^^^. 

This  substance  occurs  mixed  with  other  cresols  (ortho- 
and  meta-)  in  coal-tar.  In  its  general  properties  it  resembles 
phenol  very  closely,  though  it  melts  at  a  lower  temperature 
(36°)  and  is  far  more  difficultly  soluble  in  water  than  phenol. 
It  is  a  stronger  antiseptic  than  phenol  and  is  less  poisonous. 
When  mtroduced  into  the  animal  organism  it  appears  in  the 
urine  for  the  most  part  as  paracresyl- sulphuric  acid  (which 
also  occurs  m  horse-urine),  in  part  also  as  paraoxy-benzoic 
acid.  In  addition  to  the  paracresol  small  quantities  of  the 
other  cresols  are  also  formed  in  the  putrefaction  of  proteids. 

The  reactions  of  paracresol  in  aqueous  solution  are  very 
similar  to  those  of  phenol.  The  color  with  ferric  chloride 
solution  is,  however,  not  pure  blue,  but  a  dirty  grayish  blue. 

5.  Phenyl-acetic  Acid,  CeHgCH^CO^H. 

This  substance  crj^stallizes  in  large,  extremely  thin  leaf- 
lets which  melt  at  76.5°.  It  is  readily  soluble  in  alcohol, 
ether,  and  hot  water,  but  only  slightly  soluble  in  cold  water. 
On  oxidation  with  potassium  bichromate  and  sulphuric  acid 
it  yields  benzoic  acid.    In  the  organism  it  is  converted  into 


172  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

phenaceturic  acid,  (C6H5CH2CO)NHCH2COOH,  which  appears 
in  the  urine  and  is  found  constantly  in  horse-urine  together 
with  hippuric  acid.  Phenyl-acetic  acid  has  no  characteristic 
reactions.  It  gives  the  Liicke's  reaction  with  nitric  acid  hke 
benzoic  acid.  The  greater  solubihty  of  its  zinc  salt  ^  and  its 
conduct  in  the  organism  distinguishes  it  from  phenyl-propionic 
acid. 

6.  Phenyl-propionic  Acid,  Hydrocinnamic  Acid, 
CeHsCH^CH^COOH. 

Phenyl-propionic  acid  or  hydrocinnamic  acid  crystallizes 
in  long  fine  needles  melting  at  48.5°,  and  like  phenyl-acetic 
acid  it  yields  benzoic  acid  on  heating  with  potassium  bichro- 
mate and  sulphuric  acid.  The  solubilities  of  the  acid  are 
about  the  same  as  those  of  phenyl-acetic  acid;  the  zinc  salt 
is  very  difficultly  soluble.  In  the  organism  phenyl-propionic 
acid  is  oxidized  to  benzoic  acid,  which  appears  as  hippuric 
acid  in  the  urine.  Phenyl-propionic  acid  is  normally  the 
first  product  in  the  formation  of  hippuric  acid. 

7.  Paroxyphenyl-acetic  Acid,  C6H4<^gjj  COOH* 

This  substance  crystallizes  from  water  in  prismatic, 
extremely  brittle  needles,  usually  flat,  which  melt  at  148°. 
It  is  fairly  soluble  in  cold  water,  readily  soluble  in  hot  water, 
alcohol,  and  ether,  more  difficultly  soluble  in  benzene.  The 
aqueous  solution  gives  with  ferric  chloride  solution  a  faint 
color,  at  first  gray-violet,  then  dirty  gray.  It  reacts  positively 
with  Millon's  reagent,  and  also  gives  a  turbidity  or  precipi- 
tate with  bromine-water.  When  introduced  into  the  organ- 
ism it  is  for  the  most  part  excreted  unchanged.  A  part  is 
converted  into  oxyphenaceturic  acid. 

^  Zeitschr.  f.  physiol.  Chemie,  10,  150. 


PUTREFACTION  PRODUCTS  OF  PROTEIDS.  173 

8.  Hydroparacumaric  Acid,  Paroxyphenyl-propionic  Acid, 

^^^^XCH^CHjCO^H  (p) 

This  substance  is  verj^  similar  in  its  properties  to  the  pre- 
vious acid,  but  is  more  readily  soluble  in  water  and  in  ben- 
zene. Melting-point  127°.  According  to  Baumann  it  also 
occurs  in  the  urine,  together  with  paroxyphenyl-acetic  acid. 

9.  Skatol-carbonic  Acid,  CgHgN.COOH. 

Skatol-carbonic  acid  crystallizes  in  leaflets  which  are 
readily  soluble  in  alcohol  and  ether,  less  in  hot  water,  still 
less  in  cold  water,  and  difficultly  in  benzene.  Melting-point 
164°.  When  heated  beyond  its  melting-point  it  decomposes 
into  skatol  and  carbon  dioxide.  When  Introduced  into  the 
organism  it  is  excreted  unchanged. 

REACTIONS   OF  THE    AQUEOUS   SOLUTION    (1   :  1000).^ 

(a)  If  a  few  drops  of  pure  nitric  acid  of  1.2  specifio- gravity 
be  added  to  the  solution  and  then  some  drops  of  a  2  per  cent, 
solution  of  potassium  nitrite,  it  turns  cherry-red  after  a  time, 
then  becomes  turbid,  depositing  a  red  colormg  matter  which 
is  not  identical  with  the  nitrosoindol  nitrate. 

(6)  If  an  equal  volume  of  hydrochloric  acid  (of  1.12 
sp.  gr.)  be  added  to  the  solution  and  then  a  few  drops  of  a 
weak  (1-2  per  cent.)  solution  of  chloride  of  lime,  it  gradually 
turns  purple-red  and  deposits  a  purple-red  precipitate. 

(c)  If  some  drops  of  hydrochloric  acid  and  a  few  drops  of 
very  dilute  ferric  chloride  are  added  to  the  solution  and  the 
mixture  heated,  it  turns  cherry-red  even  before  the  tempera- 
ture reaches  the  boiling-point. 

*  Zeitschr.  f.  physiol.  Cheniie,  9,  24. 


174:  PHYSIOLOGICAL  AND  PATHOLOGICAL  CHEMISTRY. 

CH2COOH 
10.  Succinic  Acid,    | 

CH2COOH 

Succinic  acid  crystallizes  in  colorless,  quadrilateral  nee- 
dles, melting  at  182°.  It  is  fairly  readily  soluble  in  water, 
more  difficultly  in  alcohol,  and  difficultly  soluble  in  ether. 

KEACTIONS. 

1.  Heated  in  a  hard-glass  tube  closed  at  one  end,  the  acid 
melts  and  sublimes,  behig  partly  converted  into  succinic 
acid  anhydride. 

2.  When  heated  on  platinum-foil,  it  volatilizes,  giving  off 
vapors  which  have  an  extraordinarily  strong  power  to  pro- 
voke coughing. 

3.  If  some  neutral  lead  acetate  is  added  to  the  aqueous 
solution,  it  remains  clear  at  first,  but  if  the  solution  is  warmed 
gently  and  the  mixture  shaken,  lead  succinate  separates  as 
a  heavy  crystalline  precipitate. 


QUANTITATIVE   ANALYSIS. 


I. 

QUANTITATIVE    ANALYSIS    OF    SOME   INORGANIC 
COMPOUNDS. 

I.  Determination  of  the  Sulphuric  Acid  in 
Copper  Sulphate,  CuSO^+SHjO. 

Grind,  some  perfectly  pure  copper  sulphate  in  a  mortar, 
dry  it  by  pressing  between  filter-paper,  weigh  off  exactly 
about  0.5  g.  and  place  in  a  beaker  (weigh  a  cork-  or  glass- 
stoppered  weighing-tube  approximately — to  centigrams — 
introduce  about  0.5  g.,  then  weigh  accurately,  shake  out  the 
contents  of  the  tube  into  a  beaker  and  weigh  agam).  Dis- 
solve in  100  cc.  of  water,  acidify  with  a  few  drops  of  hydro- 
chloric acid,  and  heat  on  the  wire  gauze  till  it  begins  to  boil. 
Heat  to  boiling  about  10  cc,  of  barium  chloride  solution, 
acidified  with  a  drop  of  hydrochloric  acid,  and  cautiously 
add  it  to  the  copper  sulphate  solution.  Heat  the  mixture 
on  the  water-bath  until  the  barium  sulphate  has  completely 
settled,  filter  through  an  ash-free,  thin  filter  of  9  cm.  diameter 
(Schleicher  and  Schiill's,  No.  590,  or  Dreverhoff's  "ash-free 
baryta-filter-paper,"  No.  400  or  No.  412),  and  collect  the 
precipitate,  without  any  loss  on  the  filter  with  the  aid  of  warm 
water  and  a  glass  rod,  the  end  of  which  is  covered  with  a  piece 

175 


176  QUANTITATIVE  ANALYSIS. 

of  pure  rubber  tubing.  Catch  the  filtrate,  which  must  be  per- 
fectly clear,  in  a  clean  beaker,  then  wash  the  precipitate  with 
hot  water  until  the  last  wash-water  is  no  longer  made  turbid 
by  silver  nitrate  solution.  Dry  the  filter  with  its  contents 
(after  it  has  been  filled  once  with  alcohol  and  once  with  ether), 
place  it  in  a  platinum  or  porcelain  crucible,  put  the  cover 
partly  on  and  heat,  at  first  gently  and  then  more  strongly, 
until  the  contents  of  the  crucible  appear  pure  white.  Let 
cool  in  the  desiccator,  weigh,  heat  once  more,  cool,  and  weigh 
again.  Heating  with  the  blast-lamp  is  not  advisable.  Make 
two  determinations.  233.46  parts  by  weight  of  barium  sul- 
phate correspond  to  80  parts  of  SO3.  Copper  sulphate  con- 
tains 32.03  per  cent.  SO3. 

2.  Determination  of  Copper  in  Copper  Sulphate  as 
CuPRic  Oxide. 

Dissolve  about  0.8  to  1  g.  (accurately  weighed)  in  80  to 
100  cc.  of  water  in  a  porcelain  dish,  heat  until  it.  begins  to 
boil,  take  away  the  flame  and  add,  with  constant  stirring, 
dilute  sodium  hydroxide  solution  to  alkaline  reaction.  Con- 
tinue the  heating  on  the  water-bath  or  cautiously  on  the  wire 
gauze  until  the  precipitate  has  become  entirely  black,  let 
settle,  decant  off  the  supernatant  fluid  through  an  ash-free 
filter,  cover  the  copper  oxide  with  water,  heat  again,  let  set- 
tle, decant  again,  etc.,  and  repeat  this  operation  once  or  twice 
more.  Finally  place  the  entire  precipitate  on  the  filter, 
wash  thoroughly  with  hot  water  (the  last  wash-water  must 
not  become  turbid  when  treated  with  hydrochloric  acid  and 
barium  chloride  solution),  dry,  and  heat  the  precipitate, 
together  with  the  filter,  in  an  open  porcelain  crucible,  at  first 
gently  and  then  strongly.  In  case  some  of  the  copper  oxide 
sticks  so  firmly  to  the  dish  that  it  cannot  be  removed  by  the 
glass  rod,  dissolve  it  in  a  few  drops  of  nitric  acid,  evaporate 
the   solution   on  the  water-bath  in  the  porcelain  crucible 


INORGANIC  COMPOUNDS.  177 

which  is  to  be  used  for  the  determination,  and  heat  the  residue 
to  red  heat.     Copper  sulphate  contains  31.87  per  cent,  of  CuO. 

3.  Determination  of  Water  of  Crystallization  in 
Copper  Sulphate. 

Place  in  a  porcelain  crucible  a  known  weight — about  0.5 
to  0.6  g. — of  copper  sulphate,  weigh  accurately,  heat  for 
some  hours  in  an  air-bath  to  110-115°,  and  continue  heating 
at  this  temperature  until  a  constant  weight  is  obtained. 
Copper  sulphate  loses  four  molecules  of  its  water  of  crys- 
tallization at  this  temperature,  i.e.,  28.8  per  cent. 

4.  Determination  of  Calcium  in  calcium  carbonate, 
CaCOj,  AS  Calcium  Oxide. 

Place  0.3-0.4  g.  (accurately  weighed)  of  pure  calcium 
carbonate,  previously  gently  heated,  in  a  150-  to  200-cc. 
beaker,  cover  wnth  water,  dissolve  by  cautiously  adding 
dilute  hydrochloric  acid  (the  beaker  should  be  covered  with 
a  watch-glass,  which  is  afterwards  to  be  rinsed  off),  and 
dilute  with  water,  so  that  the  beaker  is  at  most  one-third 
filled  (or  the  carbonate  may  be  dissolved  in  a  flask,  the  solution 
poured  into  a  beaker,  and  the  flask  well  rinsed).  Heat  the 
solution  in  the  beaker  on  the  wire  gauze  until  it  begins  to  boil, 
and  add  about  15  cc.  of  ^.mmonium  oxalate  solution.  If  a 
precipitate  forms,  it  is  dissolved  by  adding  hydrochloric  acid. 
Then,  in  any  case,  add  ammonia  to  distinctly  alkaline  reac- 
tion, and  after  several  hours  of  standing — best  till  next  day — 
filter,  wash  the  precipitate  thoroughly  with  warm  water  (the 
last  wash-water  should  give  no  turbidity  with  nitric  acid  and 
silver  nitrate),  dr}',  and  incinerate.  Finally  heat  strongly 
with  the  blast-lamp  for  at  least  five  minutes.  After  weigh- 
ing repeat  the  heating  with  the  blast-lamp,  and  heat  even  a 
third  time  until  the  weighings  agree.  Test  the  calcium 
oxide  obtained  for  calcium  carbonate:    when  covered  with 


178  QUANTITATIVE  ANALYSIS. 

water  it  must  dissolve  on  the  addition  of  hydrochloric  acid 
without  evolving  any  gas.  Calcium  carbonate  contains  56 
per  cent,  of  calcium  oxide. 

5.  Determination  of  Aluminium  in  Potash  Alum, 
A1K(  804)2+121120,  AS  Aluminium  Oxide. 

Dissolve  about  1  g.  of  potash  alum  in  150  cc.  of  water  in 
a  porcelain  dish,  add  20  cc.  of  ammonium  chloride  solution, 
heat  till  boiling  just  begins,  and  add  ammonia  to  faintly 
alkaline  reaction  (instead  of  ammonium  chloride  and  ammonia, 
hydrochloric  acid  and  ammonia  may  also  be  used).  Heat 
for  some  time,  imtil  the  ammonia  is  for  the  most  part  driven 
off,  decant  the  supernatant  liquid  through  an  ash-free 
filter,  wash  a  few  times  by  decantation,  bring  the  entire  pre- 
cipitate on  the  filter,  wash  with  hot  water  until  the  wash- 
water  no  longer  gives  the  reaction  for  chlorides,  pour  the 
filter  full  of  alcohol,  then  full  of  ether.  After  these  have 
evaporated,  dry  completely  by  heating  in  the  air-bath  and 
incinerate,  at  first  very  cautiously  with  the  cover  on  the 
crucible,  then  heat  for  at  least  five  minutes  more  with  the 
blast-lamp,  etc.,  etc.     Potash  alum  contains  10.75  per  cent. 

of  AI2O3. 

6.  Determination  of  Chlorine  in  Sodium  Chloride, 

NaCl. 

Weigh  accurately  between  0.2  and  0.3  g.  of  pure  sodium 
chloride  (previously  gently  heated),  place  in  a  beaker, 
dissolve  in  about  100  cc.  of  water,  acidify  with  a  few  drops 
of  nitric  acid,  heat  on  the  wire  gauze,  but  not  to  boiling, 
then  add  silver  nitrate  solution  as  long  as  a  precipitate  forms. 
Continue  the  heating  on  the  water-bath  until  the  precipitate 
has  well  settled,  decant  off  the  fluid  through  an  ash-free  thin 
filter  ^  of  9  cm.  diameter,  wash  with  hot  water  to  which  a  few 

^  Use  a  Gooch  crucible  here  and  the  method  given  on  page  185. — O. 


INORGANIC  COMPOUNDS.  179 

drops  of  nitric  acid  have  been  added,  and  then  bring  the  whole 
precipitate  on  the  filter.  Wash  thoroughly  with  hot  water 
(test  the  wash-water  wdth  hydrochloric  acid),  and  dry  the 
filter  with  its  contents.  Shake  out  the  perfectly  dry  silver 
chloride  as  completely  as  possible  on  a  piece  of  black  glazed 
paper  and  cover  it  with  a  funnel.  Place  the  folded  filter  in 
a  weighed  porcelain  crucible,  heat  at  first  gently  and  then 
until  the  carbon  is  completely  burned,  let  cool,  drop  on  the 
ash  in  the  crucible  one  to  two  drops  of  nitric  acid  (with  a 
pipette),  then  one  to  two  drops  of  hydrochloric  acid,  and 
evaporate  the  acids  with  extreme  caution  (water-bath  or 
gently  heated  sand-bath,  etc.).  Heat  somewhat  more 
strongly,  let  cool  a  little  and  add  the  silver  chloride  from  the 
glazed  paper,  carefully  avoiding  any  loss  (by  using  a  brush 
or  feather  to  remove  the  last  traces  of  chloride).  Heat  the 
crucible  gently  till  the  chloride  just  begins  to  melt,  let  cool, 
and  weigh.  143.38  parts  of  silver  chloride  correspond  to 
35.45  of  chlorine.  Sodium  chloride  contains  60.59  per  cent, 
of  chlorine. 

In  order  to  remove  the  silver  chloride  from  the  crucible 
fill  it  half-full  of  dilute  sulphuric  acid,  put  in  a  piece  of  zinc, 
and  let  stand  till  next  day.  The  silver  chloride  is  reduced 
to  metallic  silver,  which  may  easily  be  removed  from  the 
crucible. 


II. 

ANALYSIS  OF  THE  URINE. 

I.  Determination  of  urea  according  to  liebig.^ 

(a)  Preparation  of  the  Solution. 

Dissolve  43  g.  of  yellow  oxide  of  mercury  (hydrargyrum 
oxydatum  flavum  via  humida  paratum,  the  red  oxide  can- 
not be  used)  in  a  mortar  in  a  mixture  of  100  cc.  of  nitric  acid 
and  the  same  amoimt  of  water.  Place  the  solution  together 
with  the  mercuric  oxide,  which  still  remains  undissolved,  in 
an  evaporating-dish,  heat  on  the  water-bath,  evaporate  to  a 
thin  sirup,  let  cool,  and  dilute  gradually  with  small  portions 
of  water  to  a  volume  of  550  cc,  let  stand  tiU  next  day  and 
filter  through  a  dry  filter. 

(b)  Standardizing  the  Solution. 

For  this  purpose  an  exactly  2  per  cent,  solution  of  urea  is 
required.  Test  the  purity  of  the  purest  possible  urea  that 
can  be  obtained.  The  aqueous  solution  should  not  give  any 
turbidity  with  nitric  acid  and  silver  nitrate  solution,  nor  with 
hydrochloric  acid  and  barium  chloride  solution;  it  should 
give  no  ammonia  when  boiled  with  a  solution  of  sodium  car- 

^  This  method,  which  gives  approximately  the  total  amomit  of  nitro- 
gen in  the  urine,  expressed  as  urea,  is  regarded  by  many  as  out  of  date. 
Moreover,  it  cannot  be  compared  with  the  Kjeldahl  method  in  accuracy. 
It  is,  however,  simpler  than  the  Kjeldahl  determination  and  does  not 
necessitate  a  complete  laboratory,  as  the  mercury  solution  can  be  bought 
and  only  needs  to  be  tested.     It  is  therefore  stiU  a  valuable  method. 

180 


THE  URINE.  181 

bonate.     If  the  urea  does  not  satisfy  these  tests  it  must  be 
recrystallized  from  absolute  alcohol. 

Moreover,  the  urea  must  in  any  case  be  freed  from  the 
adhering  hygroscopic  water  or  tested  as  to  its  complete  dry- 
ness. To  do  this,  place  about  2.5  g.  of  urea  on  a  large  watch- 
glass,  weigh  this  accurately  with  the  watch-glass,  put  into 
the  desiccator,  and  weigh  again  after  twenty-four  hours.  If 
the  weight  remains  constant  (a  difference  of  0.5  mg.  may  be 
neglected),  the  urea  may  be  at  once  used;  if  not,  put  the 
watch-glass  with  the  urea  back  into  the  desiccator  and  weigh 
again  after  twenty-four  hours.  Weigh  off  accurately  on  a 
previously  weighed  watch-glass  exactly  2  g.  of  this  urea, 
then  shake  it  into  a  funnel  placed  in  a  100-cc.  measuring- 
flask,  avoiding  any  loss,  and  rinse  off  the  watch-glass  carefully 
into  the  funnel  with  water  from  the  wash-bottle.  Wash  the 
urea  m  the  funnel  into  the  flask  with  distilled  water,  rinse  the 
funnel  several  times,  dissolve  by  cautious  shaking  the  urea 
which  still  remains  in  the  flask,  fill  the  flask  up  to  the  mark 
with  distilled  water,  put  in  the  stopper,  and  mix  thoroughly. 

When  the  urea  solution  is  prepared,  place  the  mercury 
solution  in  a  burette  (in  case  this  is  not  dry  it  is  washed  out 
several  times  vnih.  small  quantities  of  the  mercury  solution), 
and  read  the  position  of  the  fluid  in  the  burette.  Place  10  cc. 
of  the  urea  solution  in  a  small  beaker,  run  in  at  first  17-18  cc. 
of  the  mercury  solution  in  a  continuous  stream,  and  test  the 
mixture  to  determine  if  it  already  contains  an  excess  of 
mercury.  For  this  purpose  place  a  drop  of  the  well-stirred 
mixture  in  a  watch-glass  filled  with  sodium  carbonate  solu- 
tion. The  watch-glass  should  rest  upon  black  paper,  and  the 
drop  should  be  allowed  to  flow  in  from  the  side.  If  a  distinctly 
yellow  color  is  perceptible  alongside  of  the  white  mercury- 
urea  compound,  the  end-point  has  been  reached.  If  not,  add 
about  0.3  to  0.5  cc.  more  of  the  mercury  solution  and  test 
again,  etc. 


182L  QUANTITATIVE  ANALYSIS. 

The  first  determination  is  only  approximate;  it  must  be 
repeated  several  times.  It  is  very  important,  as  Pfliiger  has 
shown,  to  add  at  once,  as  nearlj^  as  possible,  the  number  of 
cubic  centimeters  of  the  mercury  solution  which  are  necessary 
to  complete  the  reaction.  The  mean  of  the  several  deter- 
minations is  taken  as  correct.  As  a  rule,  the  mercury  solu- 
tion is  too  strong  and  must  be  diluted.  The  amount  of 
water  necessary  to  be  added  (x)  is  calculated  from  the 
proportion 

a:20  —  a=v:x; 

v(20-a) 

x  = , 

a 

in  which  v  is  the  volume  of  the  mercury  solution  to  be  diluted 
and  a  the  number  of  cubic  centimeters  used.  Twenty  cubic 
centimeters  of  the  mercury  solution  will  then  correspond  to 
0.2  g.  of  urea. 

(c)  Determination  of  Urea  in  Urine. 

As  the  urine  always  contains  phosphoric  acid  and  as 
phosphoric  acid  is  also  precipitated  by  the  mercury  solution, 
it  is  necessary  in  making  a  determination  of  urea  in 
urine  by  this  method  to  remove  the  phosphoric  acid.  This 
is  done  by  mixing  50  cc.  of  the  urine,  accurately  meas- 
ured, with  25  cc.  of  Liebig's  baryta  mixture  (2  volumes  of 
baryta-water  and  1  volume  of  barium  nitrate  solution)  and 
filtering  through  a  dry  filter  into  a  dry  vessel.  Measure  off 
15  cc.  of  the  filtrate,  which  correspond  to  10  cc.  of  the 
urine.  The  titration  is  performed  in  the  same  way  as  with 
the  urea  solution.  In  regard  to  the  addition  of  the  mercury 
solution,  the  specific  gravity  serves  as  a  guide  in  the  case  of 
normal  urines.  The  number  of  cubic  centimeters  of  the 
mercurv  solution  to  be  added  at  first  is  the  same  as  the  last 
two  figures  of  the  specific  gravity  (10  cc.  if  the  specific  grav- 


THE    URINE.  183 

ity  is  1.010).  The  number  of  cubic  centimeters  of  the  mer- 
cury solution  used  to  obtain  the  end-point  of  the  reaction 
expresses  the  amount  of  the  urea  in  the  urine  in  grams  per 
hter.  If  considerably  less  than  30  cc.  is  found  necessary  to 
obtain  the  end-point,  a  correction  (according  to  Liebig)  must 
be  applied.  The  difference  between  30  and  the  number  of 
cubic  centimeters  actually  used  is  divided  by  5.  This  num- 
ber represents  the  tenths  of  cubic  centimeters  which  must 
be  subtracted  from  the  number  of  cubic  centimeters  actually 
used.  The  entire  determination  is  to  be  made  at  least 
tvnce  on  the  same  urine.  It  is  also  advisable  to  make 
a  determination  with  a  fever  urine,  in  which  case  the 
detection  of  the  end-reaction  is  more  difficult,  and  the 
greater  concentration  of  the  urine  may  also  cause  difficul- 
ties (in  the  case  of  very  concentrated  urine  take  an  equal 
quantity  of  urine  and  the  baryta  mixture ;  15  cc.  of  the  filtrate 
then  correspond  to  7.5  cc.  of  the  urine;  or  the  urine  may  previ- 
ously be  diluted) .  A  determination  of  urea  should  also  be  made 
in  a  urine  containing  albumin,  after  removing  the  albumin. 

Removal  of  Albumin  from  Urine. 

One  hundred  cubic  centimeters  of  urine  are  heated  to  boil- 
ing in  a  porcelain  dish,  the  reaction  being  kept  very  faintly 
acid  during  the  heating;  if  the  urine  is  not  acid,  then  add  cau- 
tiously a  few  drops  of  acetic  acid.  The  albumin  then  coagu- 
lates completely  in  large  flakes.  Boil  gently  for  a  few  minutes, 
let  cool,  pour  the  fluid  into  a  100-cc.  measuring-flask,  carefully 
avoiding  any  loss,  rinse  out  the  dish  with  a  small  quantity  of 
water,  so  that  the  volume  -will  not  exceed  100  cc,  let  cool 
completely  (place  in  water),  fill  up  to  the  mark  with  water, 
and  filter  through  a  dry  filter. 


Liebig's  method  gives  approximately  the  total  amount 
of  nitrogen   in  the  urine  expressed  as  urea,  but  with  an 


184  QUANTITATIVE  ANALYSIS. 

indeterminate  error  due  to  the  presence  of  sodium  chloride 
in  the  urine.  This  reacts  with  the  mercuric  nitrate  to  form 
mercuric  chloride  and  sodium  nitrate.  It  is  customary,  in 
order  to  diminish  this  error,  to  subtract  a  certain  quantity 
from  the  amount  of  mercury  solution  used,  1  cc.  for  dilute, 
1.5  cc.  for  concentrated  urines  (so-called  correction  for  salt), 
but  this  correction  is  entirely  arbitrary. 

The  above-described  simple  method,  which  is  sufficient 
for  the  physician,  has  been  very  much  improved  by  Pfliiger; 
since,  however,  the  description  of  Pfliiger's  method  would 
require  too  much  space,  the  reader  is  referred  to  the  original 
work  of  Pfliiger  or  to  the  larger  text-books. 

2.  Direct  Determination  of  Nitrogen  in  the  Urine 

ACCORDING  to   THE  KjELDAHL  METHOD. 

This  method  consists  in  the  conversion  of  all  the  nitroge- 
nous substances  in  the  urine  into  ammonium  sulphate,  car- 
bon dioxide,  and  water  by  heating  them  with  concentrated 
sulphuric  acid.  Then  add  an  excess  of  sodium  hydroxide, 
distil  off  the  ammonia  into  a  receiver  containing  a  definite 
quantity  of  an  acid  of  known  strength  (standard  acid),  and  de- 
termine by  means  of  a  standard  solution  of  ammonia  that  part 
of  the  acid  which  has  not  been  neutralized  by  the  ammonia. 

It  is  best  to  use  a  fifth-normal  solution  of  hydrochloric 
acid  and  a  tenth-normal  solution  of  ammonia.  These  are 
prepared  as  follows : 

Preparation  of  the  Fifth-normal  Solution  of  Hydrochloric  Acid. 

Dilute  19  cc.  of  pure  concentrated  hydrochloric  acid, 
specific  gravity  1.19,  to  1100  cc.  and  mix  thoroughly.  To 
25  cc.  of  this  dilute  acid  add  2  cc.  of  strong  nitric  acid,  specific 
gravity  1.2,  and  then  a  slight  excess  of  a  dilute  solution  of 
silver  nitrate.  An  excess  of  silver  nitrate  may  be  shown  to 
be  present  by  allowing  the  precipitated  silver  chloride  to 


THE   URINE.  185 

settle  and  then  adding  a  few  drops  more  of  the  silver  nitrate 
solution  to  the  clear  fluid,  when  no  further  precipitate  of  sil- 
ver chloride  will  be  formed.  Heat  to  boiling  with  constant 
stirring,  and  continue  the  boiling  for  several  minutes.  Decant 
the  clear  liquid  through  a  Gooch  crucible,  and  wash  the  pre- 
cipitate by  decantation  with  200  cc,  of  hot  water  contain- 
ing 8  cc.  of  nitric  acid  (sp.  gr.  1.2)  and  2  cc.  of  a  1  per  cent, 
silver  nitrate  solution.  During  the  washing  by  decantation 
add  the  w^ash-water  in  small  portions  and  break  up  the  pre- 
cipitate with  a  glass  rod.  Finally  transfer  the  precipitate 
completely  to  the  Gooch  crucible,  wash  with  200  cc,  of  cold 
water  and  then  with  25  to  30  cc.  of  95  per  cent,  alcohol. 
Dry  at  145-150°  and  weigh.  Continue  the  heating  at  this 
temperature  until  the  weight  remains  constant.  From 
the  weight  of  the  silver  chloride  obtained  calculate  the 
equivalent  in  hydrochloric  acid,  as  shown  in  the  following 
example : 

Twenty-five  cubic  centimeters  of  the  dilute  hydrochloric 
acid  gave  as  the  mean  of  two  duplicate  analyses  0.7751  g.  AgCl. 
Molecular  weight  of  AgCl  =  143.38.  AgCl  :  HCl  :  :  0.7751  •  x. 
Molecular  weight  of  HCl  =  36.46,     143.38  :  36.46  :  :  0.7751  :  x; 

x  =  0.1971g.  HCl  in  25  cc. 

:^  =  0.007884  g.  HCl  in  1  cc,  or  7.884  g.  HCl  per  hter.     This 

may  be  converted  into  N/5  hydrochloric  acid  containing 
7.292  g.  (I  of  36.46)  of  HCl  per  liter,  as  follows: 

7.292  :  1000  ::  7.884  ;x; 
2;  =  1081.  lee. 

x-lOOO  cc.  =  81.1  cc,  the  amount  of  distilled  water  to  be 
added  to  exactly  one  liter  of  the  dilute  hydrochloric  acid 
(containing  7.884  g.  HCl  per  liter)  to  make  it  fifth-normal 
hydrochloric  acid  (containing  7.292  g.  HCl  per  liter). 


186  QUANTITATIVE  ANALYSIS. 

Preparation  of  the  Tenth-normal  Ammonia  Solution. 

Dilute  8  cc.  of  a  concentrated  solution  of  ammonia,  specific 
gravity  0.9,  to  1100  cc.  Determine  the  strength  of  this  dilute 
solution  by  titrating  against  10  cc.  portions  of  the  standard 
N/5  hydrochloric  acid,  using  a  dilute  solution  of  cochineal  * 
as  the  indicator. 

Example:  10  cc.  of  the  standard  N/5  HCl  required  18.8 cc. 
of  the  NHg  solution  to  exactly  neutralize  it  (mean  of  two 
duplicate  determinations). 

NH3+HC1  =  NH4C1.  10  cc.  N/5  HCl  contain  0.07292  g.  HCl. 

17.07  36.48  JJCl  NH3 

.-.  36.46  :  17.07  :  :  0.07292  :x; 
x  =  0.03414  g.  NH3  in  18.8  cc. 

X 

7^-^  =  0.001816  g.  NH3  in  each  cubic  centimeter,  or 
1.816  g.  of  NH3  per  liter.  . 

This  may  be  made  exactly  a  tenth-normal  solution  contain- 
ing 1.707  g.  of  NHg  per  liter,  as  follows: 

1.707  :  1000  :  :  1.816  :  x;    x  =  1063.9  cc. 
.-.  a;-1000cc.  =  63.9cc. 

Hence  by  adding  63.9  cc.  of  distilled  water  to  exactly  one 
liter  of  the  dilute  ammonia  solution  (found  to  contain  1.816 
g.  NH3  per  liter)  the  1063.9  cc.  of  ammonia  solution  result- 
ing will  be  exactly  tenth-normal. 

Ten  cubic  centimeters  of  the  fifth-normal  hydrochloric  acid 
solution  should  be  exactly  neutralized  by  20  cc.  of  the  tenth- 
normal ammonia  solution,  using  cochineal  as  the  indicator. 

1  This  solution  of  cochineal  is  prepared  by  digesting  3  grams  of  pow- 
dered cochineal  in  a  mixture  of  50  cc.  of  strong  alcohol  and  200  cc.  of 
distilled  water  for  a  day  or  two  at  ordinary  temperature.  During  the 
digestion  the  mixture  should  be  frequently  shaken.  The  filtered  solution 
is  employed  as  the  indicator. 


THE  URINE.  187 

Determination   of   the   Total   Nitrogen   in   Urine. 
(Kjeldahl  Method.) 

1.  Apparatus.  A  Kjeldahl  digestion-flask  (250  cc),  an 
Erlenmeyer  flask  (1  liter),  two  accurate  Scheflbach  burettes, 
a  Reitmeyer  bulb-tube,  a  Liebig's  condenser,  and  the  neces- 
sarj^  stands. 

2.  Reagents.  Pure  concentrated  sulphuric  acid,  specific 
gravity  1.84,  an  alcoholic  solution  (10  per  cent.)  of  phenol 
phthalein,  a  strong  solution  of  sodium  hydroxide  free  from 
carbonates,  pure  crystallized  copper  sulphate  (CuSO^+SHsO), 
pure  crystallized  potassium  sulphate,  a  fifth-normal  solution 
of  hydrochloric  acid,  a  tenth-normal  solution  of  ammonia,  a 
dilute  solution  of  cochineal,  and  some  granulated  zinc. 

3.  The  Digestion.  Place  0.7  to  3.5  g.  of  the  substance 
(5  cc.  of  urine  or  milk  accurately  measured  from  a  burette), 
accurately  weighed,  in  a  250-cc.  Kjeldahl  digestion-flask, 
add  20  cc.  of  pure  concentrated  sulphuric  acid,  specific  gravity 
1.84,  and  about  half  a  gram  of  crystallized  copper  sulphate. 
Place  the  flask  in  an  inclined  position  on  a  stand  in  the  hood 
and  heat  over  a  low  flame  until  frothing  ceases  and  the  black- 
ened material  begins  to  wash  down  the  sides  of  the  flask. 
Lower  the  flame  and,  when  the  contents  of  the  flask  have 
cooled  somewhat,  add  from  a  20-cc.  test-tube  10  g.  of  crys- 
tallized potassium  sulphate.  Raise  the  heat  gradually  till 
the  acid  boils  briskly,  and  continue  to  boil  the  solution  till 
the  acid  becomes  clear  and  has  a  pale-blue  or  green  color. 
Then  let  cool. 

4.  The  Distillation.  When  the  contents  of  the  digestion- 
flask  are  cold  add  cautiously  about  100  cc.  of  distilled  water 
and  transfer  the  mixture  by  means  of  a  10-cm.  funnel  to  a 
one-liter  Erlenmeyer  flask.  Rinse  the  digestion-flask  three  or 
more  times  with  50  cc.  portions  of  distilled  water,  so  as  to 
make  the  contents  of  the  distillation-flask  about  350-400  cc, 


188  QUANTITATIVE  ANALYSIS. 

add  a  few  drops  of  an  alcoholic  solution  of  phenol  phthalein 
and  a  piece  of  granulated  zinc.  Connect  the  flask  by  means 
of  a  two-hole  rubber  stopper  with  a  Reitmeyer  bulb-tube 
attached  to  a  Liebig  condenser  and  with  a  bent  safety-tube. 
Attach  to  the  other  end  of  the  condenser  a  delivery-tube 
reaching  to  the  bottom  of  a  250-cc.  Erlenmeyer  flask,  which 
serves  as  a  receiver.  Place  a  carefully  measured  quantity  of 
the  N/5  hydrochloric  acid  (for  5  cc.  of  urine  25  cc,  of  the  acid) 
in  the  receiver  with  a  little  of  the  cochineal  solution  and,  if 
necessary,  add  a  little  water,  so  that  the  end  of  the  delivery- 
tube  dips  below  the  surface  of  the  liquid.  Then  add  through 
the  funnel-tube  sufficient  of  the  strong  sodium  hydroxide 
solution  to  make  the  contents  of  the  distillation-flask  dis- 
tinctly alkaline.  Mix  the  contents  of  the  flask  during  this 
addition  by  means  of  a  gentle  rotary  motion.  Then  distil 
off  about  150  cc.  of  the  liquid  and  titrate  the  contents  of 
the  receiver  with  the  N/10  ammonia  solution. 

Example:  5  cc.  of  urine,  specific  gravity  1.027,  w;ere  taken, 
25  cc.  of  N/5  acid  placed  in  the  receiver  required  6.2  cc.  of 
the  N/10  anmionia  to  completely  neutralize  after  the  distilla- 
tion. 

25  cc.  of  N/5  HC1  =  50  cc.  of  N/10  NH3. 

.*.  50  cc— 6.2  cc.=43.8  cc.  NH3  obtained  from  the  urine. 
43.8X0.001404 


5X1.027 


=  1.197%  N  in  the  urine. 


III.  Determination  of  Uric  Acid. 

This  determination  is  based  on  the  precipitation  of  the 
uric  acid  in  the  presence  of  magnesium  salts  by  an  ammonia- 
cal  silver  solution  as  silver  magnesium  urate,  and  the  solu- 
bility of  silver  chloride  in  ammonia.  To  200  cc.  of  the  urine, 
whose  specific  gravity  must  not  exceed  1020  (if  it  is  more 


THE   URINE. 


189 


concentrated  it  must  be  correspondingly  diluted),  in  a  meas- 
uring-cylinder, add  50  cc.  of  magnesia  mixture,  to  precipitate 
the  phosphoric  acid,  then  dilute  with  water  to  300  cc.  and 
filter  at  once  through  a  dry  filter  into  a  dry  vessel.    Measure 
off  200  cc.  of  the  filtrate  and  add  10  to  15  cc.  of  a  3  per  cent, 
solution  of  silver  nitrate.     The  precipitate  must  be  flocculent 
and  gelatinous;    if  it  appears  white,  it  contains  too  much 
silver  chloride,  then  some  ammonia  must  be  added  and  the 
hquid  well  stirred.     Single  white  spots  of  silver  chloride  in 
the  precipitate  do  no  harm;    they  are  without  influence  on 
the  accuracy  of  the  uric  acid  determination.     Let  the  pre- 
cipitate settle,  take  out  a  small  portion  of  the  supernatant, 
liquid  ^\ith  a  pipette,  place  it  in  a  test-tube,  and  acidify  with 
nitric  acid.     The  fluid  must  become  cloudy  from  the  forma- 
tion of  silver  chloride— an  indication  that  an  excess  of  silver 
is  present.     If  it  does  not  do  this,  make  the  test-fluid  alkalme 
again  with  anmionia,  pour  it  back  into  the  fluid  containing 
the  precipitate,  and  then  add  a  few  cubic  centimeters  more  of 
the  silver  solution.     Sometimes  it  is  also  necessary  to  add  some 
more  ammonia.     Let  the  precipitate  settle  again  and  repeat 
the   test.     Now  filter  off   the  precipitate  through  an  ordi- 
nary smooth  filter   (e.g.,  Schleicher  and    Schiill,  No.  597), 
and  carefully  remove  the  precipitate    sticking  to  the  glass 
by  means  of  a  glass  rod  and  water,  so  that  none  of  it  is 
lost.     The  precipitate  is  washed  on  the  filter  with  water 
until  a  portion  of  the  filtrate,  when  acidified  with  nitric  acid, 
remains  clear  (absence  of  silver)  and  also  shows  only  a  very 
slight   turbidity   when   silver   nitrate   is   afterwards   added 
(small  amount  of  chlorides).     Now  place  the  funnel  in  a  400- 
500-cc.  flask,  pierce  the  filter,  and  carefully  wash  the  precipi- 
tate into  the  flask  and  shake  thoroughly.     The  volume  of 
the  mixture  should  amount  to  about  200-250  cc.     Acidify 
with  a  few  drops  of  hydrochloric  acid,  pass  in  hydrogen  sul- 
phide, shaking  frequently,  until  the  fluid  is  saturated,  then 


190  QUANTITATIVE  ANALYSIS. 

heat  just  to  boiling,  filter,  rinse  out  the  flask  with  hot  water, 
and  wash  the  precipitate  a  few  times  with  hot  water.  ^  The 
filtrate  must  be  entirely  clear  and  colorless.  If  it  is  quite 
dark-colored,  it  must  at  once  be  poured  back  on  the  filter 
before  beginning  the  washing,  and  this  must  be  repeated 
imtil  it  runs  through  clear  or  almost  clear.  If  only  a  very 
little  silver  sulphide  has  passed  through,  this  may  be  tem- 
porarily neglected.  Evaporate  the  filtrate  first  over  a  free 
flame,  then  on  the  water-bath  to  a  few  cubic  centimeters,  add 
about  five  to  eight  drops  of  hydrochloric  acid,  and  let  stand 
till  next  day:  the  uric  acid  crystallizes  and  is  usually  only 
slightly  colored. 

If  any  silver  sulphide  separates  during  the  evaporation, 
it  may  be  filtered  off.  It  is  advisable,  however,  to  do  this 
at  a  time  when  the  volume  of  the  fluid  has  not  been  reduced 
more  than  one-half,  as  otherwise  loss  from  the  precipitation 
of  the  uric  acid  may  occur.  It  is  now  necessary  to  deter- 
mine the  quantity  of  the  uric  acid.  For  this  purpose  dry  a 
small  filter  in  a  watch-glass  apparatus  or  in  a  weighing-tube 
(open)  at  110-115°,  and  weigh  the  whole  apparatus  (closed). 
Bring  the  entire  amount  of  the  uric  acid  on  the  filter,  using 
a  part  of  the  filtrate  for  the  rinsing.  When  all  the  uric  acid 
is  on  the  filter,  wash  with  a  small  quantity  of  water  until  a 
portion  of  the  filtrate  ceases  to  give  any  turbidity  with  nitric 
acid  and  silver  nitrate  solution.  The  filtrate  and  the  wash- 
water  are  collected  and  measured.  If  possible,  the  volume 
of  the  filtrate  and  wash-water  should  not  amount  to  more 
than  50-60  cc.  Then  wash  twice  with  absolute  alcohol  and 
once  with  ether,  place  the  filter  in  the  watch-glass  apparatus 
or  in  the  weighing-tube,  dry  (open)  and  weigh  (closed). 
The  difference  between  the  two  weights  is  uric  acid. 

In  calculating  the  results  it  is  usual  to  apply  a  correction 

^  It  is  always  advisable  to  examine  the  silver  sulphide  under  the  micro- 
scope to  detect  any  uric  acid  that  may  be  mixed  with  it. 


THE   URINE.  191 

for  the  solubility  of  the  uric  acid ;  however,  this  is  permissible 
onl}'  when  the  quantity  of  uric  acid  is  not  abnormally  small 
and  the  quantity  of  the  filtrate  and  wash-water  does  not 
exceed  60  cc.  It  is  customary  to  add  0.5  mg.  of  uric  acid 
for  every  10  cc.  of  the  filtrate  plus  the  wash-water.  The 
product  of  the  amount  obtained  by  0.75  gives  the  percentage 
of  uric  acid  in  the  urine. 

The  above-described  method,  though  exact,  is  undeniably 
involved  and  somewhat  difficult  to  carry  out.  The  Hop- 
kins method,  in  which  the  uric  acid  is  precipitated  as  the 
ammonium  salt  and  then  titrated,  gives  apparently  just  as 
exact  results.  According  to  Folin  ^  the  follo\ving  is  the  best 
procedure : 

To  100  cc.  of  the  urine  (according  to  Womer  it  is  best  to 
heat  the  urine  previously  to  40-45°  C.)  add  20  to  30  g.  of 
powdered  ammonium  chloride  or  30  g.  of  ammonium  sulphate, 
and  dissolve  this  by  shaking.  Filter  off  the  precipitate  after 
two  hours  and  wash  free  from  chlorine  with  a  concentrated 
solution  of  ammonium  sulphate  and  rinse  into  a  flask  with 
hot  water.  When  cold  add  15  cc.  of  concentrated  sulphuric 
acid  and  titrate  to  a  permanent  rose-color  with  one-twen- 
tieth normal  potassium  permanganate  solution  (1.581  g. 
potassium  permanganate  to  one  liter;  the  solution  is  to  be 
tested  as  to  its  correctness  with  one-twentieth  normal  oxalic 
acid  or  with  iron  ammonium  sulphate).  The  temperature 
of  the  fluid  titrated  should  be  60°  to  63°.  If  it  is  higher, 
wait  until  it  cools  to  this  temperature.  The  number  of  cubic 
centimeters  of  potassium  permanganate  used  multiplied  by 
3.75  ^  gives  the  quantity  of  the  uric  acid  in  milligrams. 

Womer  '  recommends  the  following  procedure  for  the 
determination:    150  cc.  of  the  urine  are  heated  in  a  beaker 

'  Zeitschr.  f.  physiol.  Chemie,  24,  224. 

*  Ibid. 

» Ibid.  29,  70. 


192  QUANTITATIVE  ANALYSIS. 

to  40-50°  and  30  g.  of  ammonium  chloride  dissolved  therein. 
The  precipitate  of  ammonium  urate  is  filtered  off  after  stand- 
ing for  one-half  to  one  hour  and  washed  free  from  chlorine 
with  a  10  per  cent,  ammonium  sulphate  solution;  then  it  is 
dissolved  on  the  filter  in  hot  1  to  2  per  cent,  sodium  hydroxide 
solution,  the  filter  washed  with  hot  water,  and  the  filtrate 
and  wash-water  heated  in  a  porcelain  dish  on  the  water-bath 
until  all  the  anmionia  has  been  driven  off.  The  alkaline 
uric  acid  solution  is  rinsed  into  a  Kjeldahl  flask,  stirred  up 
with  15  cc.  of  concentrated  sulphuric  acid,  some  copper  sul- 
phate and  potassium  sulphate  added,  and  the  ammonia 
resulting  estimated  in  the  usual  way. 

One  cubic  centimeter  of  the  fifth-normal  hydrochloric 
acid  corresponds  to  8.4  mg.  of  uric  acid.  The  heating  of 
the  alkaline  uric  acid  solution  may  also  be  done  in  a  large' 
Kjeldahl  flask,  but  then  constant  attention  is  necessary  since 
loss  may  easily  occur  from  foaming.  In  this  case  the  entire 
determination  may  be  carried  to  completion  in  the  same 
flask. 

iv.  determination  of  creatinine  as  creatinine  zinc 

Chloride. 

In  this  determination  we  proceed  exactly  as  in  the  method 

given  for  the  detection  of  creatinine,  see  page  102,  using  80 

cc.   of  the  alcohoUc  filtrate  for  the  precipitation  with  zinc 

chloride.     The    creatinine  zinc  chloride  which   separates  is 

collected  on  a  dried  and  weighed  filter,  as  in  the  case  of  uric 

acid,  washed  with  alcohol  until  the  wash-liquid  no  longer  gives 

the  reaction  for  chlorides,  dried  and  weighed.     The  amount 

obtained  when  multiplied  by  0.39  gives  the  percentage  of 

creatinine.     (One  hundred  parts  of  creatinine  zinc  chloride 

0.6242x5 
correspond  to  62.42  of  creatinine,  hence  — — 3 — —  =  in  round 

numbers  0.39.) 


THE   URINE  193 

V.  Determination  of  Ammonia. 

Place  in  the  crystallizing-dish  of  the  Schlosing's  apparatus 
25  cc.  of  filtered  urine  and  in  the  porcelain  dish  of  the  same 
apparatus  10  cc.  of  fifth-normal  acid.  Then  add  to  the 
urine  about  25  cc.  of  milk  of  lime  (one  part  by  weight 
of  calcium  hydroxide  shaken  with  twelve  parts  of  water)  and 
quickly  put  on  the  cover.  After  forty-eight  to  seventy-two 
hours  wash  out  the  contents  of  the  upper  dish  into  a  beaker, 
mix  thoroughly,  and  titrate  with  tenth-normal  ammonia  solu- 
tion. The  difference  corresponds  to  the  ammonia  evolved 
from  the  urine.  One  cubic  centimeter  of  fifth-normal  acid 
=  0.003-414  g.  NHg.  If  25  cc.  of  urine  were  used  and  fifth- 
normal  acid,  then  the  product  of  the  difference  in  cubic 
centimeters  by  0.013656  gives  the  quantity  of  ammonia  in 
100  cc.  of  urine.  Test  any  water  which  has  condensed  on  the 
top  or  walls  of  the  apparatus  for  an  alkaline  reaction.  If  it 
reacts  alkaline,  rinse  the  apparatus  with  water  and  titrate  it 
also. 

VI.  Determination  of  Urea. 

(a)  According  to  Morner  and  Sjoqvist.  Mix  in  a  flask 
5  cc.  of  urine,  5  cc.  of  baryta  mixture  (10  g.  barium  chloride, 
3-4  g.  barium  hydroxide,  and  100  cc.  of  water),  and  add 
100  cc.  of  a  mixture  of  alcohol  and  ether  (two  volumes  of  97 
per  cent,  alcohol  and  one  volume  of  ether) .  Let  stand  till  next 
day,  filter,  wash  with  the  alcohol-ether  mixture,  evaporate 
at  a  gentle  heat,  and  when  the  volume  has  reached  25  cc. 
add  some  water  and  some  milk  of  magnesia  (one  part  of 
burnt  magnesia  and  twelve  parts  of  water),  and  heat,  to 
drive  out  ammonia,  till  the  vapor  no  longer  reacts  alkaline. 
Then  wash  the  fluid  together  with  the  precipitate  into  a 
Kjeldahl  flask,  add  some  dilute  sulphuric  acid,  then  10  cc. 
of  the  concentrated  acid,  determine  the  nitrogen  as  usual 


194  QUANTITATIVE  ANALYSIS. 

and  calculate  it  as  urea.  Not  applicable  to  urines  rich  in 
hippuric  acid.^  The  heating  with  magnesia  may  also  be 
entirely  omitted  if  the  ammonia  be  determined  separately 
and,  calculated  as  urea,  be  deducted  from  the  amount  of 
urea  found. 

(h)  According  to  Freund  and  Topfer.^  Five  cubic  centi- 
meters of  urine  are  treated  with  the  same  volume  of  alcohol 
and  evaporated  on  the  water-bath  to  dryness,  the  residue 
ground  and  extracted  several  times  with  absolute  alcohol  and 
filtered  into  a  Kjeldahl  flask.  The  alcohol  is  then  driven 
off  as  completely  as  possible  on  the  water-bath,  about  70 
cc.  of  a  saturated  ethereal  solution  of  oxalic  acid  are  poured 
into  the  flask,  and  the  precipitate  formed  is  allowed  to  settle. 
Filter  the  solution,  leaving  as  much  of  the  precipitate  as 
possible  in  the  flask,  and  wash  with  60  to  80  cc.  of  ether  in 
several  portions. 

When  the  ether  has  evaporated  from  the  filter  the  con- 
tents of  the  filter  are  washed  into  the  fiask  with  distilled  water 
and  the  solution  of  the  contents  of  the  flask  is  titrated  with 
fifth-normal  sodium  hydroxide  solution,  using  phenol  phtha- 
lein  (two  drops  of  a  1  per  cent,  solution)  as  an  indicator. 
Afterwards  the  determination  of  nitrogen  according  to 
Kjeldahl  is  undertaken. 

VII.  Determination  of  Oxalic  Acid. 

The  determination  of  oxalic  acid  is  made  according  to 
the  method  given  for  the  detection  of  this  substance,  page 
104,  but  the  extraction  with  ether  must  be  made  five  times. 
Collect  the  calcium  oxalate  without  loss  on  an  ash-free  filter, 
wash,  dry,  place  in  a  weighed  crucible,  heat  with  the  blast- 
lamp,  to  convert  the  calcium  oxalate  into  calcium  oxide 

1  Salaskin  and  Zaleski,  Zeitschr.  f.  physiol.  Chemie,  28,  73. 

2  Wiener  klin.  Rundschau,  1899,  No.  23. 


THE  URINE.  195 

(,CaO),  and  weigh.  This  weight  multipHed  by  1.61  gives  the 
quantity  of  the  oxahc  acid  (C2H2O4).  After  the  weighing, 
dissolve  the  hme  in  a  little  dilute  nitric  acid :  there  should  be 
no  evolution  of  carbon  dioxide.  The  solution  obtained  is 
tested  with  ammonium  molybdate  for  phosphoric  acid. 

Vin.  Determination  of  Phenol  or  Cresol. 

Proceed  as  directed  in  the  qualitative  detection  of  these 
substances,  page  108.  Add  bromine-water  to  the  distillate 
until  a  permanent  yellow  color  results,  let  stand  a  few  days, 
filter  through  a  weighed  filter  which  has  been  dried  in  a  desic- 
cator over  sulphuric  acid,  dry  in  the  dark  in  the  desiccator 
over  sulphuric  acid  until  the  weight  is  approximately  con- 
stant, and  weigh.  331  parts  of  the  precipitate  correspond 
to  94  parts  of  phenol  or  108  of  cresol. 

The  object  of  letting  the  precipitate  stand  is  for  the  pur- 
pose of  converting  the  tetrabromcresol,  C6H(CH3)Br30Br, 
which  first  forms  into  tribromphenol.* 

IX.   DETERMINATION  OF  ALBUMIN. 

One  hundred  cubic  centimeters  or,  in  case  of  urines 
containing  a  large  amount  of  albumin,  only  50  cc.  of  the 
previously  filtered  and  perfectly  clear  urine  is  placed  in  a 
beaker,  which  should  be  only  half-filled  by  the  liquid,  a 
drop  of  acetic  acid  is  added  in  case  the  reaction  is  not  dis- 
tinctly acid,  and  the  mixture  is  heated  for  half  an  hour  in  the 
water-bath,  so  that  the  beaker  is  surrounded  by  the  water, 
until  the  coagulum  becomes  coarsely  flocculent.  The  water- 
bath  should  not  be  too  hot  in  the  beginning.  If  the  albumin 
does  not  become  coarsely  flocculent,  add  a  few  more  drops 
of  acetic  acid.      Filter  through  a  filter  dried  at  110-115° 

*  For  the  titration  method  of  Kossler  and  Penny  for  determining 
phenol  see  C.  Neuberg,  Zeitschr.  f.  physiol.  Chemie,  28,  123. 


196  QUANTITATIVE  ANALYSIS. 

and  which  is  not  too  small,  bring  the  albumin  completelj^  on 
the  filter  with  the  aid  of  a  glass  rod,  wash  with  hot  water 
until  a  portion  of  the  wash-water  no  longer  gives  the  reaction 
for  chlorides,  pour  the  filter  full  of  absolute  alcohol  t-wice, 
then  twice  full  of  ether,  dry  at  110-115°  to  constant  weight, 
and  weigh.  If  the  quantity  of  the  albumin  is  considerable,  it  is 
necessary  to  incinerate  the  filter  and  albumin  and  to  deduct 
the  weight  of  the  ash  found  from  the  weight  of  the  albumin. 
In  this  case,  of  course,  an  ash-free  filter  must  be  used. 

X.  Determination  of  Glucose. 

Two  principal  methods  are  in  use,  the  determination 
by  means  of  the  optical  rotation  and  the  reduction  of  cupric 
oxide  to  cuprous  oxide  in  alkaline  solution.  For  practice 
in  the  determination  of  glucose,  first  use  a  3  to  4  per  cent, 
solution  of  glucose  and  then  a  diabetic  urine  or  a  3  to  4 
per  cent,  solution  of  glucose  in  urine. 

(a)  Determination  by  Means  of  Polarized  Light. 

Before  using  the  polarization  apparatus  the  correct  posi- 
tion of  the  zero-point  must  be  determined.  All  readings 
must  be  noted  in  figuring  the  mean.  The  filling  of  the 
tube  is  done  as  follows :  First  wash  it  out  with  distilled  water, 
then  two  or  three  times  with  the  solution  to  be  analyzed. 
This  precaution  is  absolutely  essential.  If  it  is  omitted, 
streaks  are  formed  in  the  fluid  owing  to  the  gradual  mixing 
of  the  sugar  solution  with  the  water  adhering  to  the  walls 
of  the  tube.  These  interfere  with  the  observation  in  exactly 
the  same  way  as  they  would  if  in  the  glass  cover-plate,  or 
lens.  After  thorough  rinsing,  place  the  tube  on  the  table, 
pour  it  full  of  sugar  solution  or  urine,  so  that  the  fluid  forms 
a  rounded  top,  and  slide  on,  from  the  side,  the  weU-cleaned 
cover-plate,  so  that  all  air-bubbles  are  excluded.  Then  screw 
on  the  brass  cap  moderately  tight.     The  cover  must  not 


THE   URINE.  197 

be  screwed  on  too  tight,  since  the  glass  itself  under  very 
strong  pressure  may  become  optically  active.  With  a  new 
tube  the  cap  is  usually  only  lightly  screwed  on.  A  series 
of  readings  is  always  made  and  the  numbers  obtained  noted ; 
sometimes  single  observations  fall  entirely  out  of  the  series; 
these  may  be  stricken  out  without  hesitation. 

The  tube  must  be  carefully  cleaned  immediately  after 
using  and  also  be  rinsed  with  distilled  water.  This  is  espe- 
cially important  in  the  examination  of  urine.  In  putting  the 
apparatus  away  the  cover  is  only  screwed  on  loosely  m  order 
that  the  rubber  ring  may  not  stick  to  the  glass. 

The  urine  must  be  perfectly  clear  and  should  always 

be  filtered :  further,  it  must  not  be  too  strongly  colored.     If 

it  is  not  possible  to  get  it  perfectly  clear  by  filtration  or  if  it 

is  too  strongly  colored,  it  must  be  treated  with  precipitating 

reagents,  which  also  remove  the  coloring-matter.     Neutral 

lead  acetate  is  most  generally  used  for  this  purpose.     The 

urine  is  shaken  with  powdered  lead  acetate  in  a  dry  flask 

(to  50  cc.  of  the  urine  about  1  g.  of  the  acetate)  and  filtered 

through  a  dry  filter  into  a  dry  beaker.     If  the  urine  which 

passes  through  at  first  is  turbid,  pour  it  back  repeatedly  on 

the  filter.     Instead  of  this  method  of  procedure  we  may  also 

mix  four  volumes  of  urine  with  one  volume  of  a  saturated 

lead  acetate  solution  and  filter  through  a  dry  filter.     The 

dilution  must,  of  course,  be  taken  into  consideration  in  cah 

culating  the  result.     If   the  urine  contains  oxybutyric  acid 

— which  is  always  to  be  assumed  when  it  contams  aceto- 

acetic   acid— a   correction   must   be  applied  to  the  number 

read  for  the  amount  of   sugar,  owing  to  the  Isevorotation 

caused  by  the  oxybutyric  acid.     The  urine  is  allowed  to 

ferment,  and  then  the  rotation  is  determined.     This  is  to  be 

added  to  the  rotation  caused  by  the  glucose.    Combined 

glucuronic  acid  may  also  under  certain  circumstances  cause 

lajvorotation.     (P.    Mayer,   Berl.    klin.    Wochenschr.    1900, 


198  QUANTITATIVE  ANALYSIS. 

No.  1.)     The  calculation  of  the  amount  of  glucose  is  made  as 
follows : 

(X.  X 100 

,     rr)  r  =  Number  of  grams  of  sugar  in  100  cc.  of  urine, 

in  which  a  =  observed  rotation  using  sodium  light,  ' 

Z  =  length  of  tube  in  decimeters. 

(b)  Determination  by  Means  of  Reduction. 

Under  certain  definite  conditions  one  molecule  of  glucose 
reduces  very  nearly  five  molecules  or  ten  equivalents  of 
copper  oxide  to  cuprous  oxide,  hence  180  parts  of  anhydrous 
glucose  will  reduce  the  oxide  from  1248.8  parts  of  crystallized 
copper  sulphate,  CuSO^+SHjO,  to  cuprous  oxide. 

1.  fehling's  titration  method. 

Preparation  of  the  Solutions,  (a)  34.639  g.  of  pure  copper 
sulphate  in  crystals,  which  have  not  effloresced,  are  accurately 
weighed  off  on  a  large  watch-glass,  dissolved  by  warming 
with  water  in  a  dish,  the  solution  placed  in  a  500-cc.  measur- 
ing-flask and,  after  it  is  perfectly  cold,  filled  up  to  the  mark. 

(h)  About  173  g.  of  potassium  sodium  tartrate  (Rochelle 
salts)  are  dissolved  by  warming  with  a  little  water,  the 
solution  placed  in  a  500-cc.  measuring-flask,  100  cc.  of  sodium 
hydroxide  solution  of  1.34  specific  gravity  added,  and,  after 
the  mixture  is  cold,  filled  up  to  the  volume  of  500  cc. 

Mix  equal  vohimes  of  the  two  fluids — about  25  cc.  meas- 
ured with  a  pipette — in  a  dry  beaker:  deep-blue  fluid,  Feh- 
ling's solution,  10  cc.  of  which  is  equivalent  to  0.05  g.  of 
glucose.  Test  the  solution  by  diluting  a  portion  of  it  in  a 
test-tube  with  about  four  times  its  volume  of  water  and 
heating  to  boiling:  no  cuprous  oxide  should  precipitate. 


THE   URINE.  199 

The  Determination. 

Dilute  the  sugar  solution  or  the  urine  so  that  the  fluid 
contains  about  0.5  per  cent,  of  sugar  or  somewhat  more,  and 
put  this  solution  into  a  burette.^  Then  measure  off  accu- 
rately with  a  pipette  10  cc.  of  the  Fehling's  solution  into  a 
moderately  deep  porcelain  dish  or  into  a  flask,  add  about  40 
cc.  of  water,  heat  to  boiling,  and  then  let  the  sugar  solution 
run  in.  Red  cuprous  oxide  or  yellow  cuprous  hydroxide 
very  soon  precipitates.  On  further  addition  of  the  sugar 
solution  the  precipitate  of  the  cuprous  oxide  increases,  while 
the  blue  color  of  the  solution  decreases.  It  is  now  necessary 
to  recognize  the  point  when  the  blue  color  of  the  fluid  disap- 
pears, i.e.,  when  all  the  copper  oxide  has  been  reduced  and 
yet  no  excess  of  sugar  is  present.  When  it  is  thought  that 
this  point  is  nearly  reached,  filter  a  small  quantity  of  the 
fluid,  removed  by  means  of  a  pipette,  through  a  small  filter 
of  very  close  filter-paper — the  filtrate  must  contain  no  sus- 
pended cuprous  oxide,  which  very  readily  goes  through  the 
filter — acidify  with  hydrochloric  acid  and  make  alkaline 
with  anmionia:  the  fluid  must  not  turn  blue.  If  it  does, 
then  add  0.5  cc.  more  of  the  sugar  solution,  heat  and  test 
again  for  copper,  etc.  Of  course  this  first  titration  is  always 
only  an  approximation.  If  the  first  test  shows  that  the 
solution  is  free  from  copper,  it  is  possible  that  too  much 
sugar  solution  has  been  added,  and  the  entire  determination 
must  then  be  repeated  and  the  sugar  solution  must  be  added 
more  carefully.  The  strength  of  the  dilute  sugar  solution 
in  per  cent,  is  equal  to  5  divided  by  the  number  of  cubic  cen- 
timeters used  to  complete  the  reaction. 


*  The  specific  gravity  is  to  be  used  to  determine  the  amount  of  the 
dilution ;  of  course  it  ia  not  always  possible  at  the  first  attempt  to  make 
the  correct  dilution. 


200  QUANTITATIVE  ANALYSIS. 

2.    GRAVIMETRIC  METHOD. 

Thirty  cubic  centimeters  of  Fehling's  solution  are  diluted 
with  50  cc.  of  water  and  heated  to  boiling  in  a  porcelain  dish. 
Twenty  cubic  centimeters  of  the  diluted  sugar  solution  are 
added,  the  solution  is  kept  gently  boiling  for  five  minutes, 
and  then  it  is  diluted  with  about  120  cc.  of  water  which  has 
been  previously  boiled.  The  fluid  must  remain  blue.  Filter 
through  a  dried  and  weighed  filter  (Schleicher  and  Schiill, 
No.  590,  about  9  cm.,  or  the  ash-free  so-called  baryta-filter- 
paper  of  Dreverhoff  in  Dresden),  wash  with  hot  water  until 
a  portion  of  the  wash-water  is  no  longer  made  turbid  with 
hydrochloric  acid  and  barium  chloride,  then  with  absolute 
alcohol  and  ether,  dry  at  110-115°  and  weigh.  The  differ- 
ence in  the  two  weights  is  the  cuprous  oxide.  To  calculate 
the  amount  of  sugar  from  the  cuprous  oxide  multiply  by 

18 
35;^  =  0.5028. 

More  exact  than  the  above  method,  but  also  more  difficult 
to  carry  out,  is  the  modification  according  to  Allihn.  In  this 
the  cuprous  oxide  is  collected  on  an  asbestos  filter,  reduced, 
by  heating  in  a  current  of  hydrogen,  to  metallic  copper  and 
weighed  as  such. 

Since  the  reducing  power  of  the  sugar  towards  copper 
oxide  is  somewhat  variable,  according  to  the  concentration 
of  the  sugar  solution,  it  is  not  permissible,  in  very  exact  deter- 
minations, to  calculate  the  amount  of  sugar  from  the  quan- 
tity of  copper  obtained.  In  this  case  we  must  use  an  empir- 
ically determined  table  (pages  202,  203),  which  gives  directly 
the  quantity  of  sugar  corresponding  to  the  weight  of  the 
copper  found. 

Of  course  this  table  may  also  be  used  when  the  cuprous 
oxide  itself  has  been  weighed.  It  is  only  necessary  to  calcu- 
late this  cuprous  oxide  as  copper  by  multiplying  by  318  and 


THE   URINE.  201 

dividing  by  358  (the  equivalent  weight  of  copper  is  63.6;  of 
cuprous  oxide  71.6;  and  of  cupric  oxide  79.6).  K.  B.  Leh- 
mann  ^  and  von  Riegler  ^  have  both  given  a  method  for  the 
determination  of  sugar,  wWch  consists  in  heating  the  solu- 
tion in  which  the  sugar  is  to  be  estimated  with  a  measured 
quantity  of  an  excess  of  Fehhng's  solution,  filtering  from 
the  precipitated  cuprous  oxide  (or  letting  it  settle),  and  deter- 
mining by  the  iodometric  method  of  de  Haen  the  copper 
which  remains  in  solution.  The  difference  between  the 
quantity  of  thiosulphate  used  and  the  amount  of  thiosul- 
phate  which  the  quantity  of  the  Fehling's  solution  taken 
would  require  corresponds  to  the  cupric  oxide  reduced  by 
the  sugar.  This  method  has  been  tested  and  recommended 
by  Benjamin.^  The  method  of  procedure  as  given  by  the 
two  authors  named  is  somewhat  different.  According  to 
Lehmann,  60  cc.  of  the  Fehling's  solution  is  boiled  with  25 
CO.  of  the  sugar  solution,  the  mixture  is  then  placed  in  a  250- 
cc.  measuring-flask,  and  the  flask  filled  up  to  the  mark  with 
water.  Mix  thoroughly,  filter  through  a  dry  filter  (or  let  set- 
tle), take  out  of  the  filtrate  with  a  pipette  50  cc,  make  this 
slightly  acid  with  sulphuric  acid,  add  2  to  3  g.  of  potassium 
iodide,  about  3  cc.  of  starch  paste  (1  g.  of  arrowroot  starch 
boiled  -with  100  cc.  of  water),  and  titrate  with  1/10  normal 
thiosulphate  solution  the  iodine  set  free: 

2CUSO4+ 4KI  =  2X380,+ CUJ2+ 12, 

2Na2S203+ 12  =  Na2S,0e+ 2NaI. 

Twenty  cubic  centimeters  of  Fehling's  solution    require 
27.74  cc.  of  1/10  normal  thiosulphate  solution.     One  cubic 

*  Arch,    der    Hygiene,    30,    267;    Maly's    Jahresber.  f.  Thierchemie, 
1897,  64. 

'  Zeitschr.  f.  analyt.  Chem.  37,  22. 

*  Deutsche  med.  Wochenschr.  1898,  551. 


202 


QUANTITATIVE  ANALYSIS. 


TABLE  TO  DETERMINE  THE   AMOUNT  OF  GLUCOSE: 


§ 

(a 

6 
o 

i 

i 

o 

6 

o 

o 

6 

o 

Q. 

t> 

a 

o 

a 

o 

a 

o 

a 

a 

o 

a 

o 

§" 

3 

a 

3 

a 

3 

ft 

3 

a 

3 

ft 

3 

a 

3 

6 

O 

cS 

5 

cS 

3 

6 

3 

o 

3 

O 

o 

3 

o 

3 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

10 

6.1 

43 

22.4 

76 

38.8 

109 

55.5 

142 

72.3 

175 

89.5 

208 

106.8 

11 

6.6 

44 

22.9 

77 

39.3 

110 

56.0 

143 

72.9 

176 

90.0 

209 

107.4 

12 

7.1 

45 

23.4 

78 

39.8 

111 

56.5 

144 

73.4 

177 

90.5 

210 

107.9 

13 

7.6 

46 

23.9 

79 

40.3 

112 

57.0 

145 

73.9 

178 

91.1 

211 

108.4 

14 

8.1 

47 

24.4 

80 

40.8 

113 

57.5 

146 

74.4 

179 

91.6 

212 

109.0 

15 

8.6 

48 

24.9 

81 

41.3 

114 

58.0 

147 

74.9 

180 

92.1 

213 

109.5 

16 

9.0 

49 

25.4 

82 

41.8 

115 

58.6 

148 

75.5 

181 

92.6 

214 

110.0 

17 

9.5 

50 

25.9 

83 

42.3 

116 

59.1 

149 

76.0 

182 

93.1 

215 

110.6 

18 

10.0 

51 

26.4 

84 

42.8 

117 

59.6 

150 

76.5 

183 

93.7 

216 

111.1 

19 

10.5 

52 

26.9 

85 

43.4 

118 

60.1 

151 

77.0 

184 

94.2 

217 

111.6 

20 

11.0 

53 

27.4 

86 

43.9 

119 

60.6 

152 

77.5 

185 

94.7 

218 

112.1 

21 

11.5 

54 

27.9 

87 

44.4 

120 

61.1 

153 

78.1 

186 

95.2 

219 

112.7 

22 

12.0 

55 

28.4 

88 

44.9 

121 

61.6 

154 

78.6 

187 

95.7 

220 

113.2 

23 

12.5 

56 

28.8 

89 

45.4 

122 

62.1 

155 

79.1 

188 

96.3 

221 

113.7 

24 

13.0 

57 

29.3 

90 

45.9 

123 

62.6 

156 

79.6 

189 

96.8 

222 

114.3 

25 

13.5 

58 

29.8 

91 

46.4 

124 

63.1 

157 

80.1 

190 

97.3 

223 

114.8 

26 

14.0 

59 

30.3 

92 

46.9 

125 

63.7 

158 

80.7 

191 

97.8 

224 

115.3 

27 

14.5 

60 

30.8 

93 

47.4 

126 

64.2 

159 

81.2 

192 

98.4 

225 

115.9 

28 

15.0 

61 

31.3 

94 

47.9 

127 

64.7 

160 

81.7 

193 

98.9 

226 

116.4 

29 

15.5 

62 

31.8 

95 

48.4 

128 

65.2 

161 

82.2 

194 

99.4 

227 

116.9 

30 

16.0 

63 

32.3 

96 

48.9 

129 

65.7 

162 

82.7 

195 

100.0 

228 

117.4 

31 

16.5 

64 

32.8 

97 

49.4 

130 

66.2 

163 

83.3 

196 

100.5 

229 

118.0 

32 

17.0 

65 

33.3 

98 

49.9 

131 

66.7 

164 

83.8 

197 

101.0 

230 

118.5 

33 

17.5 

66 

33.8 

99 

50.4 

132 

67.2 

165 

84.3 

198 

101.5 

231 

119.0 

34 

18.0 

67 

34.3 

100 

50.9 

133 

67.7 

166 

84.8 

199 

102.0 

232 

119.6 

35 

18.5 

68 

34.8 

101 

51.4 

134 

68.2 

167 

85.3 

200 

102.6 

233 

120.1 

36 

18.9 

69 

35.3 

102 

51.9 

135 

68.8 

168 

85.9 

201 

103.1 

234 

120.7 

37 

19.4 

70 

35.8 

103 

52.4 

136 

69.3 

169 

86.4 

202 

103.7 

235 

121.2 

38 

19.9 

71 

36.3 

104 

52.9 

137 

69.8 

170 

86.9 

203 

104.2 

236 

121.7 

39 

20.4 

72 

36.8 

105 

53.5 

138 

70.3 

171 

87.4 

204 

104.7 

237 

122.3 

40 

20.9 

73 

37.3 

106 

54.0 

139 

70.8 

172 

87.9 

205 

105.3 

238 

122.8 

41 

21.4 

74 

37.9 

107 

54.5 

140 

71.3 

173 

88.5 

206 

105.8 

239 

123.4 

42 

21.9 

75 

38.3 

108 

55.0 

141 

71.8 

174 

89.0 

207 

106.3 

240 

123.9 

THE   URINE. 


203 


FROM  THE  WEIGHT  OF  COPPER  (ALLIHN). 


a 

0. 

& 
0 

i 

o 
o 

O 

o 

0. 

a 
o 
o 

3 

o 

c 

0. 

D. 

6 

6 

o 
u 

3 

5 

c 

B. 

6 

i 

0 
o 

s 

6 

o 

a 
a 

d 

i 

o 
u 

3 

o 

0) 

ft 

i 

i 

o 
o 

3 

a 

c 

a 

a 

d 

i 

o 
t> 

3 

5 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

241 

124.4 

273;  141. 7 

305 

159.3 

337 

177.0 

369 

195.1 

401 

213.5 

433 

232.2 

242 

125.0 

274  142.2 

306 

159.8 

338 

177.6 

370 

195.7 

402 

214.1 

434 

232.8 

243 

125.5 

275  142.8 

307 

160.4 

339 

178.1 

371 

196.3 

403 

214.6 

435 

233.4 

244 

126.0 

276  143.3 

308 

160.9 

340 

178.7 

372 

196.8 

404 

215.2 

436 

233.9 

245 

126.6 

277,143.9 

309 

161.5 

341 

179.3 

373 

197.4 

405 

215.8 

437 

234.5 

246 

127.1 

278144.4 

310 

162.0 

342 

179.8 

374 

198.0 

406 

216.4 

438 

235.1 

247 

127.6 

279  145.0 

311 

162.6 

343 

180.4 

375 

198.6 

407 

217.0 

439 

235.7 

248 

128.1 

280  145.5 

312 

163.1 

344 

180.9 

376 

199.1 

408 

217.5 

440 

236.3 

249 

128.7 

281146.1 

313 

163.7 

345 

181  .5 

377 

199.7 

409 

218.1 

441 

236.9 

250 

129.2 

282  146.6 

314 

164.2 

346 

182  .1 

378 

200.3 

410 

218.7 

442 

237.5 

251 

129.7 

283  147.2 

315 

164.8 

347 

182  .6 

379 

200.8 

411 

219.3 

443 

238.1 

252 

130.3 

284147.7 

316 

165.3 

348 

183  .2 

380 

201.4 

412 

219.9 

444 

238.7 

253 

130.8 

285  148.3 

317 

165.9 

349 

183  .7 

381 

202.0 

413 

220.4 

445 

239.3 

254 

131.4 

286  148.8 

318 

166.4 

350 

184  .3 

382 

202.5 

414 

221.0 

446 

239.8 

255 

131.9 

287,149.4 

319 

167.0 

351 

18L9 

383 

203.1 

415 

221.6 

447 

240.4 

256 

132.4 

288149.9 

320 

167.5 

352 

185.  4 

384 

203.7 

416 

222  2 

448 

241.0 

257 

133.0 

289 

150.5 

321 

168.1 

353 

186.  0 

385 

204.3 

417 

222.8 

449  241.6 

258 

133.5; 

290 

151.0 

322 

168.6 

354 

186.  6 

386 

204.8 

418 

223.3 

450 

242.2 

259 

134.  ll 

291 

151.6 

323 

169.2 

355 

187  .2 

387 

205.4 

419 

223.9 

451 

242.8 

260 

134.6 

292 

152,1 

324 

169.7 

356 

187  .7 

388 

206.0 

120 

224.5 

452 

243.4 

261 

135.1 

293 

152.7 

325 

170.3 

357 

188.  3 

389 

206 . 5 

421 

225.1 

453 

244.0 

262 

135.7, 

294 

153.2 

326 

170.9 

358 

188.9 

390 

207.1 

422 

225.7 

4.54 

244.6 

263 

136.2, 

295 

153.8 

327 

171.4 

359 

189.4 

391 

207.7 

423 

226.3 

455 

245.2 

264 

136.8 

296 

154.3 

328 

172.0 

360 

190.0 

392 

208.3 

424 

226.9 

456 

245.7 

265 

137.3 

297 

1.54.9 

329 

172.5 

361 

190.6 

393 

208.8 

425 

227 . 5 

457 

246.3 

266 

137.8 

298 

155.4 

330 

173.1 

362 

191.1 

394 

209.4 

426 

228 . 0 

458 

246.9 

267 

138.4 

299 

156.0 

331 

173.7 

363 

191.7 

395 

210.0 

427 

228 .  () 

-159 

247.5 

268 

138.9 

300 

156.5 

332 

174.2 

364 

192.3 

.396 

210.6 

428 

220.2 

460 

248.1 

269 

139.5' 

301 

157.1 

333 

174.8 

365 

192.9 

397 

211.2 

429 

229.8 

461 

248.7 

270 

140.0, 

302 

157.6 

334 

175  3 

366 

193.4 

398 

211.7 

430 

230.4 

462 

249.3 

271 

140.6 

303 

158.2 

335 

175.9 

367 

194.0 

399 

212.3 

431 

231.0 

163 

249.9 

272 

141.1 

304 

158.7 

336 

176.5 

368 

194.6 

400 

212.9 

432 

231.6 

204  QUANTITATIVE  ANALYSIS. 

centimeter  of  1/10  normal  thiosulphate  solution  corresponds 
to  6.36  mg.  of  copper. 

If  the  quantity  of  cuprous  oxide  which  separates  is  very 
smaU  it  may  be  well  washed,  dissolved  in  nitric  acid,  the 
nitrous  acid  removed  by  means  of  urea,  and  the  copper 
directly  determined.  The  results  may  easily  be  too  high. 
(K.  B.  Lehmann.) 

Very  frequently  in  the  case  of  urines  containing  a  small 
amount  of  sugar  the  cuprous  oxide  does  not  precipitate. 
In  this  case  neither  the  titration  method  nor  the  gravimetric 
method  can  be  used.  For  such  cases  the  following  solu- 
tion is  recommended  by  Arthus:  125  cc.  of  Fehling's  solu- 
tion and  5  g.  of  ferrocyanide  of  potassium  are  diluted  to  one 
liter.  Eight  cubic  centimeters  of  this  solution  correspond 
to  1  cc,  of  Fehling's  solution.  This  solution  is  only  decolor- 
ized by  the  glucose ;  however,  the  recognition  of  the  end-point 
is  rendered  very  uncertain  by  the  color  of  the  mixture. 

Normal  urine  contains  reducing  substances  correspond- 
ing to  about  0.2  to  0.3  per  cent,  of  glucose.  If,  therefore,  we 
titrate  a  normal  urine  to  which  a  known  quantity  of  glucose 
has  been  added,  the  determination  will  of  course  give  results 
correspondingly  too  high.  For  diabetic  urine  this  error  is 
apparently  not  so  great  and  need  not  be  considered,  espe- 
cially when  the  urine  is  diluted. 

The  determination  of  the  sugar  from  the  volume  of  the 
carbon  dioxide  evolved  on  fermenting  the  urine  is  very  con- 
venient, though  less  accurate.  For  this  purpose  we  may 
use  the  empirically  graduated  ''Fermentation  Sacchari- 
meter "  of  Einhorn,  Fiebig,  or  Lohnstein,  the  latter  also  for 
undiluted  urine  (Allg.  med.  Centralzeitung,  1899,  No.  101, 
and  Miinch.  med.  Wochenschr.  1899,  No.  50). 


THE  URINE.  205 

XI.  DETERMINATION  OF  HYDROCHLORIC  ACID. 

Hydrochloric  acid  is  usually  expressed  in  the  analyses  as 
sodium  chloride. 

Mohr's  Titration  Method.  Principle:  If  potassium  chro- 
mate  and  then  silver  nitrate  solutions  be  added  to  sodium 
chloride  solution,  silver  cliloride  alone  precipitates.  Only 
after  all  the  chlorine  has  combined  with  the  silver  does  the 
silver  chroma te  separate.^  Tliis  mixes  wdth  the  precipitated 
silver  chloride  and  imparts  to  it  an  orange  color. 

The  silver  solution  is  advantageously  prepared,  so  that 
1  cc.  corresponds  to  0.01  g.  of  sodium  chloride.  This  is 
obtained  by  dissolving  29.054  g.  of  pure  fused  silver  nitrate 
in  one  liter  of  water  or  7.2635  g.  in  250  cc.  (For  the  method 
of  making  up  the  solution  see  ''Determination  of  Urea," 
page  181.) 

Determination.  To  10  cc.  of  the  urine,  in  a  porcelain  dish 
or  in  a  flask  placed  on  white  paper,  add  100  cc.  of  water 
and  then  a  few  drops  of  a  solution  of  potassium  chromate 
until  a  distinct  yellow  color  results.  Then  let  the  silver  solu- 
tion run  in  from  a  burette  until  the  red  color,  which  forms 
where  the  silver  solution  runs  in,  no  longer  disappears  when 
the  liquid  is  thoroughly  stirred.  The  first  trace  of  a  perma- 
nent orange  color  marks  the  end-point  of  the  reaction.  The 
first  titration  gives  only  an  approximate  result.  The  deter- 
mination is  to  be  repeated  once  more  with  the  same  urine. 

Xn.   DETERMINATION  OF  THE  TOTAL  SULPHURIC  ACID. 

Heat  100  cc.^  of  filtered,  perfectly  clear,  urine  in  a  beaker 
on  the  wire  gauze  to  boiling  with  10  cc.  of  hydrochloric  acid. 
Keep  boiling  gently  for  about  ten  minutes,  then  remove  the 

'  The  silver  phosphate  precipitates  after  the  chromate. 

'  In  the  case  of  concentrated  urines  50  cc.4-  50  cc.  of  water  are  snfScient. 


206  QUANTITATIVE  ANALYSIS. 

flame,  and,  after  a  few  minutes,  add  cautiously  10-15  cc.  of 
bariimi  chloride  solution  which  has  been  previously  heated. 
Then  let  stand,  preferably  till  next  day,  so  that  the  barium 
sulphate  may  settle  completely.  If  this  does  not  take  place, 
heat  the  beaker  on  the  water-bath  until  the  barium  sulphate 
has  settled  and  the  fluid  appears  perfectly  clear.  Filter,  after 
the  heating  on  the  water-bath,  through  a  small,  ash-free,  close 
filter  of  9  cm.  diameter,  and  transfer  the  precipitate  completely 
to  the  filter  with  the  aid  of  a  glass  rod  having  a  piece  of  rub- 
ber tubing  on  its  end.  The  filtrate  must  be  perfectly  clear. 
If  it  is  not,  it  is  made  so  by  pouring  it  repeatedly  back  on  the 
filter.  Test  the  clear  filtrate  by  means  of  dilute  sulphuric 
acid,  to  determine  if  sufficient  barium  chloride  has  been 
added,  then  wash  the  precipitate  with  warm  water  until  a 
portion  of  the  last  wash-water  is  no  longer  rendered  turbid 
vdth  silver  nitrate  solution,  pour  the  filter  full  of  absolute 
alcohol  once  or  twice  to  remove  coloring  matters  (indigo 
blue  and  red)  and  to  dry,  and  then  once  full  of  ether.  To 
determine  the  quantity  of  the  barium  sulphate  thus  obtained 
place  the  filter,  which  is  perfectly  dry  after  some  minutes, 
together  with  the  precipitate,  in  a  weighed  platinum  cruci- 
ble, heat  gently  at  first,  with  the  cover  partly  on,  and  then 
more  strongly  for  about  five  minutes  or  longer  (with  thick 
paper),  at  any  rate  until  the  contents  of  the  crucible  appear 
perfectly  white,  let  cool  and  weigh.  The  difference  in  weight 
gives  the  quantity  of  barium  sulphate.      This  weight  mul- 

98  08 
tiplied  by  ^^^^-^t.  =  0.4201  gives  the  quantity  of  sulphuric  acid, 

and  multiplied  by  ^»»  .^  =  0.34293  the  quantity  of  sulphuric 
anhydride. 


THE   URINE.  207 

Xni.  Determination  of  the  Total  Sulphur  and  of  the 
Neutral  Sulphur. 

Fift}'  cubic  centimeters  of  the  urine,  in  the  case  of  con- 
centrated urines  25  cc,  are  evaporated  in  a  platinum  dish  on 
the  water-bath  to  a  small  volume,  20  g.  of  nitrate  mixture 
(3  parts  of  saltpeter  to  1  part  of  sodium  carbonate)  are 
added  and  heated  cautiously  from  the  side  till  the  mass  fuses 
completely  and  becomes  white.  On  account  of  the  sulphur 
present  in  illmninating-gas  a  spirit-flame  is  preferable  for  the 
heating.  When  cold  dissolve  the  fused  mass  in  water,  rinse 
it  into  a  flask,  and  cautiously  run  into  the  flask,  very  gradu- 
ally through  a  funnel,  100  cc.  of  hydrochloric  acid.  Then 
heat  on  the  sand-bath,  with  the  funnel  in  the  neck  of  the 
flask,  until  the  evolution  of  gas  has  completely  ceased,  place 
in  a  porcelain  dish,  evaporate  to  dryness,  pour  on  100  cc.  of 
hydrochloric  acid,  stirring  thoroughly,  again  evaporate  to 
■drjmess,  and  repeat  this  operation  once  more.  Take  up  the 
dry  residue  with  water,  filter  (to  remove  silicic  acid)  into  a 
beaker,  heat  on  the  wire  gauze  till  boiling  begins,  and  add 
cautiously  10  cc.  of  hot  bariiun  chloride  solution.  Filter 
next  day,  etc.,  as  in  the  determination  of  the  total  sulphuric 
acid. 

By  subtracting  the  amount  of  sulphur  in  the  total  sul- 
phuric acid  from  the  total  sulphur  we  obtain  the  neutral 
sulphur. 

XIV.  Determination  of  Ethereal  Sulphuric  Acid. 

For  this  determination  it  is  best  to  use  urine  voided  after 
the  use  of  phenol  or  the  urine  of  persons  suffering  with  ileus. 

Mix  equal  volumes  (75  or  100  cc.)  of  urine  and  alkaline 
barium  chloride  solution  (mixture  of  two  volumes  of  baryta- 
water  and  one  volume  of  barium  chloride  solution)  in  a  dry 
beaker,  stir  thoroughly,  and  filter  after  a  few  minutes  through 


208  QUANTITATIVE  ANALYSIS. 

a  dry  filter  into  a  dry  vessel.  Measure  off  100  cc.  of  the  clear 
filtrate  (on  standing  the  filtrate  becomes  cloudy  from  the 
formation  of  barium  carbonate),  acidify  very  faintly  with 
hydrochloric  acid,  then  add  10  cc.  more  of  hydrochloric  acid 
and  proceed  as  directed  in  the  determination  of  the  total 
sulphuric  acid,  only  with  the  difference  that  the  further 
addition  of  barium  cliloride  solution  is  unnecessary. 

XV.  Determination  of  Phosphoric  Acid. 

Phosphoric  acid  is  generally  determined  by  titration  with 
a  solution  of  uranium  acetate.  If  a  solution  of  uranium  (or 
uranyl)  nitrate  or  acetate  be  added  to  a  solution  of  secondary 
sodium  phosphate  acidified  with  acetic  acid  and  containing 
sodium  acetate,  a  yellowish-white  precipitate  of  uranyl  phos- 
phate is  formed  according  to  the  equation  ^ 

U02(N03)2+Na2HP04  =  U02HPO,+2NaN03. 

Any  excess  of  uranium  is  readily  detected :  a  drop  of  the 
mixture  then  gives,  with  a  drop  of  potassium  ferrocyanide 
solution,  a  brownish-red  precipitate  of  uranyl  ferrocyanide. 
This  is  the  so-called  end-reaction;  it  appears  only  when  the 
phosphoric  acid  is  completely  precipitated  and  a  slight  excess 
of  uranium  is  present.  Instead  of  this  some  cochineal  solu- 
tion may  be  added  to  the  solution  of  the  phosphates  =  an 
excess  of  uranium  produces  a  green  color,  but  only  when  the 
fluid  contains  no  free  nitric  acid.  There  is  nothing  to  pre- 
vent using  both  end-reactions  at  the  same  time. 

Preparation  of  the  Uranium  Solution. — Dissolve  about 
33  g.  of  commercial  sodium  uranate  by  warming  with  about 
200  cc.  of  water  and  the  smallest  possible  quantity  of  nitric 

^  As  the  urine  contains  monosodium  phosphate  the  equation  is 
U02(N03)2+  NaH^PO,  =  U02HP0,+  NaN03+  HNO3. 
The  sodium  acetate  is  added  to  get  rid  of  the  nitric  acid  set  free  in  the 
reaction. 


THE   URINE.  209 

acid,  and  dilute  to  1100  cc.  The  strength  of  tliis  solution 
must  be  empirically  determined  by  titration  with  a  solution 
of  sodium  phosphate  of  known  strength.  Weigh  out  accu- 
rately 10.0944  g.  of  pure,  dry  sodimii  phosphate,  Na2lIP04+ 
I2H2O,  which  has  not  effloresced,  dissolve  in  water,  and  fill 
up  to  one  liter.  The  preparation  of  this  solution  is,  however, 
often  ver}^  difficult,  sometimes,  indeed,  even  impossible,  as 
the  sodium  phosphate  effloresces  in  dry  air.  It  is  better, 
therefore,  to  proceed  as  follows :  About  12  g.  of  sodium  phos- 
phate are  dissolved  in  1100  cc.  of  water;  50  cc.  of  the  well- 
mixed  solution  are  evaporated  in  a  platinum  or  porcelain 
dish,  dried  and  ignited.  The  residue,  consisting  of  sodium 
pyrophosphate,  Na4P207,  must  weigh  0.1875  g.  If  it  weighs 
more,  the  solution  must  be  correspondingly  diluted.  Fifty 
cubic  centimeters  of  this  solution,  measured  off  with  a  pipette, 
are  run  into  a  beaker,  5  cc.  of  acetic  acid  mixture  (100  g. 
sodium  acetate,  100  cc.  of  dilute  acetic  acid  Ph.  G.  Ill  diluted 
to  one  hter)  added,  then  a  few  drops  of  cochineal  tincture,  and 
heated  almost  to  boiling.  Now  run  in  about  18  cc.  of  the 
uraniimi  solution  and  observe  the  color  of  the  mixture.  At 
the  same  time  take  out  a  drop  of  the  solution  with  a  glass  rod 
and  bring  it  into  contact  with  a  drop  of  potassium  ferrocya- 
nide  solution  (it  is  ad\asable  to  have  a  number  of  drops  of 
the  ferrocyanide  arranged  in  series  on  a  white  porcelain  plate). 
If  a  faint-brown  color  appears  after  a  few  moments,  the  end- 
reaction  is  attained  (the  mixture  or  the  precipitate  will  then 
also  show  a  greenish  color).  If  the  brown  coloration  does 
not  appear,  let  more  of  the  uranium  solution  run  in  and  test 
each  time,  after  the  addition  of  0.2  cc.  When  the  end-point 
is  reached  heat  the  mixture  for  some  minutes  and  test  again 
for  the  end-reaction,  etc.  If  the  solution  is  of  the  correct 
strength,  50  cc.  of  the  sodium  phosphate  solution  will  require 
20  cc.  of  the  uranium  solution.  Usually  this  is  not  the  case, 
but  less  is  required.     If,  for  example,  19.4  cc.  were  used. 


210  QUANTITATIVE  ANALYSIS. 

then  we  must  add  to  every  19.4  cc.  0.6  cc.  of  water.  Twenty- 
cubic  centimeters  of  this  uranium  solution  will  then  corre- 
spond to  0.1  g.  P2O5. 

The  determination  of  the  phosphoric  acid  in  the  urine  is 
made  exactly  as  described  above  in  the  titration  and  also 
with  50  cc.  The  number  of  cubic  centimeters  of  the  uranium 
solution  used  divided  by  10  gives  the  amount  of  phosphoric 
acid,  P2O5,  in  grams  for  one  liter  of  urine. 


III. 

ANALYSIS  OF  THE  F^CES. 

The  fseces,  advantageously  collected  in  a  weighed  dish 
(weighed  with  a  glass  rod  for  stirring),  are  dried  by  long-con- 
tinued heating  on  a  water-bath,  stirring  frequently  with  the 
glass  rod,  until  they  appear  dry  enough  to  powder.  The 
drj'ing  may  be  facilitated,  according  to  Poda,^  by  repeatedly 
pouring  on  absolute  alcohol  (after  four  to  six  hours  add  50  cc. 
of  absolute  alcohol,  then  after  another  hour  25  cc,  and  heat 
again).  Weigh  the  dish  and  determine  thus  the  weight  of 
the  air-dried  fseces.  These  are  then  quickly  powdered  and 
preserved  in  a  well-closed,  glass-stoppered  bottle.  The  loss 
due  to  the  removal  of  the  contents  of  the  dish  and  powdering 
makes  no  difference. 

1.  Determination  of  the  Amount  of  Water.  Weigh  about 
1.5  to  2  g.  in  a  cork-  or  glass-stoppered  weighing-tube 
(10  to  15  cm.  long)  whose  weight  is  known  to  a  centigram, 
shake  out  the  contents  of  the  tube  into  a  weighed  platinum 
dish,  weigh  the  tube  again,  and  thus  determine  the  weight 
of  the  quantity  used.  Heat  at  110°  until  the  weight  is  con- 
stant. When  required  the  amount  of  water  may  be  calcu- 
lated from  the  moist  substance. 

2.  The  Determination  of  the  Ash  is  made  with  the  same 
material.  Heat  the  residue  obtained  in  the  determina- 
tion of  water  cautiously  until  it   ceases  to  give  off  vapor, 

'  Zeitschr.  f.  physiol.  Chem.  26,  355. 

211 


212  QUANTITATIVE  ANALYSIS. 

then  ignite  completely  by  heating  more  strongly.  If  the 
complete  oxidation  to  ash  cannot  be  accomplished  in  this 
way,  extract  the  residue  on  the  water-bath  with  water, 
filter  through  a  thin  ashless  filter,  on  which  the  ash  contain- 
ing carbon  is  also  brought,  so  far  as  this  may  be  done  without 
difficulty.  Rinse  out  the  dish,  and  filter  several  times  with 
hot  water  (of  course  rinsing  water  is  used  for  washing  the 
filter),  and  dry  the  filter  with  its  contents.  Dry  the  platinum 
dish  also.  Place  the  filter  with  the  ash  containing  carbon  in 
the  platinima  dish  and  ignite.  The  oxidation  to  ash  now  takes 
place  quickly.  I^et  cool,  transfer  the  aqueous  extract  with- 
out loss  to  the  platinum  dish,  evaporate  to  dr}Tiess,  and 
ignite  the  residue.  Naturally  we  may  also  evaporate  the 
aqueous  extract  by  itself.  The  soluble  and  the  insoluble 
salts  are  thus  separately  determined. 

3.  To  Determine  the  Amount  of  Nitrogen  use  1  to  1.5 
g.  according  to  the  amount  of  nitrogen  present,  which  is 
greater  with  a  meat  diet  than  with  mixed  food.  The  exact 
determination  of  the  weight  of  the  faeces  used  is  ascertained  as 
in  the  case  of  the  determination  of  the  amount  of  water.  The 
dust  which  remains  stickhig  to  the  neck  of  the  Kjeldahl  flask  is. 
rinsed  into  the  flask  with  a  little  water,  15  cc.  of  sulphuric 
acid  and  0.5  g.  of  copper  sulphate  are  added,  and  the  flask  is 
heated,  at  first  with  a  small  flame  until  the  contents  have  a 
faint-blue  or  green  color,  etc.  If  the  oxidation  takes  place 
slowly,  it  may  be  hastened  by  the  cautious  addition  of  a  little 
finely  pulverized  permanganate.  Thirty-five  cubic  centi- 
meters of  fifth-normal  acid  are  placed  in  the  receiver.  This 
is  sufficient  for  almost  all  cases.  With  faeces  very  rich  in 
nitrogen  use  20  cc.  of  half-normal  acid  (for  the  details  of  the 
process  see  imder  Urine,  page  187). 

4.  Determination  of  the  Ether  Extract  (Fat).  Three 
or  four  grams  of  the  material  (accurately  weighed)  are 
extracted  in  a  Soxhlet  apparatus  with  ether,  the  ether  extract 


THE  FJECES.  213 

evaporated  in  a  light  weighed  Erlenmeyer  flask,  the  last 
traces  of  ether  driven  out  by  means  of  a  current  of  air  or  CO2, 
and    the   flask   heated   for   several   hours  at  80°  or  for  a 
short  time  at  105°  and  weighed.    If  it  is  desired  to  know 
also  the  quantity  of  the  fatty  acids  present  in  the  form  of 
soap,  moisten  a  few  grams  of  the  material,  accurately  weighed, 
in  a  dish  with  dilute  hydrochloric  acid  (1.3),  dry  on  the  water- 
bath,  transfer  the  residue  completely  to  the  Soxhlet  thimble, 
by  wiping  out  the  dish  with  pieces  of  filter-paper  and  placing 
these  also  in  the  thimble,  and  extract  with  ether.     More 
ether  extract  will  be  obtained  than  in  the  first  determina- 
tion.   The  excess  is  due  to  the  fatty  acids  present  in  the  form 
of  salts.     If  the  residue  has  a  brown  color,  it  is  better  to 
extract  it  once  more  with  ether.     If  the  quantity  of  the 
f^ces  is  small,  the  powder  which  has  already  been  extracted 
with  ether  may  be  used  for  the  determination  of  the  fatty 
acids,  or,  if  only  a  total  determination  of  the  fat  and  fatty 
acids  is  desired,  the  powder  may  be  dampened  with  hydro- 
chloric acid  and  dried  on  the  water-bath  before  the  extrac- 
tion. 

5.  Determination  of  the  Starch  or  of  the  Carbohydrates 
according  to  Marker.  Place  between  3  and  4  grams  of  the 
material  (accurately  weighed)  in  a  porcelain  vessel  (the  pots 
in  which  Liebig's  extract  of  beef  is  packed  are  very  suitable), 
pour  on  25  cc.  of  a  1  per  cent,  solution  of  lactic  acid  and  30 
cc.  of  water,  stir  thoroughly  with  a  glass  rod,  rinse  this  off 
with  the  smallest  possible  quantity  of  water,  and  heat  in  an 
autoclave  for  two  and  one-half  hours  at  three  atmospheres 
pressure.  By  this  means  the  starch  will  be  converted  into 
dextrin,  while  the  cellulose  will  not  be  attacked.  When 
there  is  no  longer  any  pressure  in  the  autoclave,  it  is  opened, 
the  contents  of  the  porcelain  vessel,  together  with  the  sus- 
pended matter,  are  placed  in  a  250-cc.  measuring-flask,  rins- 
ing the  vessel  well  with  water  and,  when  perfectly  cold,  the 


214  QUANTITATIVE  ANALYSIS. 

flask  is  filled  up  to  the  mark  with  water.  Let  settle  and  take 
out  200  cc.  of  the  supernatant  fluid  (using  a  100-cc.  pipette) ; 
instead  of  this  we  may  also  filter  through  a  dry  filter  and 
measure  off  200  cc.  of  the  filtrate.  Place  the  measured  fluid 
in  a  400-cc.  flask,  add  15  cc.  of  hydrochloric  acid,  and  heat 
two  and  one-half  hours  in  an  actively  boiling  water-bath,  to 
convert  the  dextrin  into  glucose;  let  cool,  place  in  a  300-cc. 
measuring-flask,  nearly  neutralize  with  caustic  soda  solu- 
tion (if  too  much  alkali  has  been  added,  make  faintly  acid 
again  with  dilute  hydrochloric  acid),  and  make  the  sugar 
determination — best  gravimetrically  with  50  cc. — or  titrate 
according  to  Fehling  (the  latter  method  is  to  be  recommended 
only  with  larger  quantities  of  starch).  Instead  of  this, 
method,  that  of  L.  Liebermann,  which  requires  no  autoclave, 
may  also  be  used.     (See  Analysis  of  Bread,  page  229.) 

6.  Determination  of  the  Total  Phosphorus.  (a)  By 
fusing  with  the  oxidizing  mixture.  About  1  to  1.3  g.  of  the 
material  (accurately  weighed)  is  fused  with  20  g.  of  the 
oxidizing  mixture.  It  is  best  to  proceed  as  follows:  About 
two-thirds  of  the  oxidizing  mixture  is  placed  in  a  smooth 
mortar^  a  hole  is  made  in  the  mixture  with  the  pestle,  the 
material  placed  in  this,  ground,  and  the  mixture  transferred 
to  the  platinum  dish.  The  mortar  is  then  rinsed  out  with 
the  rest  of  the  oxidizing  mixture  in  two  portions  and,  if  neces- 
sary, wiped  out  mth  a  feather  or  a  brush.  These  portions 
are  placed  at  the  side  of  the  dish,  and  the  heating  is  also 
begun  here  and  continued  until  all  the  organic  substance  is^ 
burned.  It  is  very  advantageous,  towards  the  end  of  the 
operation,  to  hold  the  dish  in  the  flame  with  the  tongs.  The 
complete  combustion  is  then  more  readily  accomplished. 
The  fused  mass  is  then  dissolved  in  water,  the  solution  intro- 
duced, without  loss,  by  means  of  a  funnel,  into  a  flask,  nitric 
acid  cautiously  added  to  strongly  acid  reaction  (20  to  25  cc.) 
and  heated  on  the  sand-bath,  with  a  funnel  set  in  the  neck  of 


THE  F^CES.  215 

the  flask,  until  the  evolution  of  gas  has  entirely  ceased.  The 
fluid  is  then  placed  in  a  porcelain  dish,  evaporated  to  a 
volume  of  about  100  cc,  10  g.  of  ammonium  nitrate  and 
50  cc.  of  the  ammonium  molybdate  solution  added,  and  let 
stand  at  room  temperature  or  at  60°  to  70°  till  next  day. 
Decant  through  a  small  filter,  and  wash  the  dish  and  filter  a 
few  times  with  a  solution  containing  150  g.  of  ammonium 
nitrate  and  10  cc.  of  nitric  acid  in  the  liter.  Dissolve  the 
yellow  residue  in  the  dish  in  dilute  ammonia  (1:3)  by  warm- 
ing, filter  the  solution  through  the  same  filter,  and  wash  dish 
and  filter  with  ammonia.  Now^  add  to  the  solution  con- 
tained in  a  beaker,  whose  volume  should  at  most  amount  to 
100  cc.  (preferably  less),  hydrochloric  acid  until  a  yellow  pre- 
cipitate begins  to  separate  again,  then  about  one-fourth  of 
the  volume  of  ammonia  and  10  cc.  of  magnesium  chloride  mix- 
ture. Filter  next  day,  transfer  the  precipitate  of  MgNH4P04 
with  the  help  of  the  filtrate  completely  to  the  filter,  then 
wash  with  dilute  ammonia  (1:3)  until  a  portion  of  the  filtrate 
when  acidified  with  nitric  acid  is  no  longer  rendered  turbid  ^vith 
silver  nitrate  solution  or  only  faintly,  drj",  and  ignite  strongly. 
The  magnesium  pyrophosphate  obtained  must  be  white,  or 
at  most  a  very  faint  gray.  If  it  is  not  possible  to  attain  this 
by  ignition  alone,  add  a  little  nitric  acid,  dry,  and  ignite  again. 
111.36  parts  of  MgzPjOy  correspond  to  31  parts  of  phos- 
phorus =71  parts  of  P2O5. 

(b)  Instead  of  fusing  vnth  the  oxidizing  mixture  the 
organic  matter  may  also  be  destroyed,  according  to  A.  Neu- 
mann,^ by  heating  with  sulphuric  acid  and  ammonium 
nitrate.  To  2  to  3  g.  of  the  material  take  15  cc.  of  sulphuric 
acid  and  15  g.  of  ammonium  nitrate.  As  in  the  nitrogen 
determination,  the  powdered  faeces  are  placed  in  a  Kjeldahl 
flask,  washed  down  with  a  little  water,  15  cc.  of  sulphuric 

'Chem.  Centralbl.,  1898,  1,  219. 


216  QUANTITATIVE  ANALYSIS. 

acid  added,  and  heated  till  colorless.  The  ammonium  nitrate 
is  added  in  three  portions  after  cooling  each  time.  When 
cold,  make  alkaline  with  ammonia  and  acidify  with  acetic 
acid.  In  case  no  precipitate  (of  ferric  phosphate)  is  formed, 
the  phosphoric  acid  may  be  titrated  with  the  uranium  solu- 
tion; otherwise  it  must  be  determined  gravimetrically.  We 
may  also  in  any  case  acidify  with  nitric  acid  and  precipitate 
with  ammonium  molybdate  solution. 

Pfeiffer  and  Scholz  (Deutsches  Arch.  f.  klin.  Med.  63,  373) 
recommend  to  heat  0.5  to  1.0  g.  of  the  dried  faeces  with  10  cc. 
of  sulphuric  acid  and  5  g.  of  potassium  sulphate  and  to  pre- 
cipitate the  diluted  solution  with  ammonia  and  magnesia 
mixture.  The  precipitate  is  filtered  off,  dissolved  in  dilute 
acetic  acid,  together  with  the  ammonium  magnesium  phos- 
phate clinging  to  the  beaker  (the  volume  of  the  solution 
should  not  exceed  50  cc.) ;  the  solution  is  then  neutralized 
with  caustic  soda,  5  cc.  of  acetic  acid  mixture  added  (see 
under  Urine),  and  titrated  with  the  uranium  solution  (the 
method  does  not  take  into  consideration  the  ferric  phosphate, 
which  is  seldom  entirely  absent;  the  analyses  given,  how- 
ever, show  concordant  results). 

7.  Determination  of  the  Total  Sulphur.  Fuse  about  2 
g.  with  30  g.  of  the  oxidizing  mixture,  then  proceed  as  directed 
in  the  determination  of  sulphur  in  the  urine,  page  207. 


IV. 

ANALYSIS  OF  MEAT. 

In  all  determinations  finely  chopped  meat  is  used.  Great 
■care  should  be  taken  that  the  material  used  for  the  single 
determinations  should  represent  as  far  as  possible  a  correct 
average  sample. 

1.  Determination  of  the  Amount  of  Water.  Between 
2  and  3  g.,  accurately  weighed,  are  placed  in  a  platinum  (or 
porcelain)  dish  and  dried  to  constant  weight,  at  first  on  the 
water-bath,  then  at  110°  to  115°. 

2.  Determination  of  the  Amount  of  Ash.  The  same 
sample  serves  for  the  determination  of  the  amount  of  ash. 
Cautiously  char  the  substance  at  first,  then  heat  until  vapors 
no  longer  escape,  grind  the  carbon  with  the  agate  pestle  or  a 
glass  rod,  extract  with  hot  water,  filter  through  an  ash-free 
filter,  wash  thoroughly,  and  preserve  the  filtrate.  Now  dry 
the  filter  with  the  carbon,  put  it  in  the  dish,  and  ignite  com- 
pletely. When  the  dish  is  cold  add  the  filtrate,  evaporate 
to  dryness,  dry  and  ignite.  See  "Analysis  of  the  Faeces," 
page  211. 

3.  Determination  of  the  Amount  of  Nitrogen.  The 
amount  of  nitrogen  may  be  determined  directly  in  fresh 
meat,  but  the  operation  is  not  very  easy  and  the  determina- 
tion after  drying  is  to  be  preferred.  We  proceed  best  as  fol- 
lows; A  considerable  quantity,  about  50  g.,  of  meat  is  accu- 
rately weighed  in  a  dish  together  with  a  glass  rod,  and  dried 
on  the  water-bath  until  the  meat  may  be  powdered.    Now 

217 


218  QUANTITATIVE  ANALYSIS. 

weigh  the  dish  with  its  contents,  remove  the  dry  meat  com- 
pletely with  a  spatula,  and  proceed  as  directed  in  the  method 
for  the  ' '  Determination  of  Nitrogen  in  the  Faeces/'  using  0.5 
g.  of  the  material  and  35  cc.  of  the  fifth-normal  acid.  From 
the  value  obtained  the  percentage  of  nitrogen  in  the  fresh 
meat  is  calculated.  From  this  the  amount  in  the  dried  meat 
may  be  estimated  by  making  use  of  the  determination  of  the 
amount  of  water  in  the  fresh  meat  (see  1  above). 

There  is  no  reason  why  the  determination  of  the  amounts 
of  water  and  ash  may  not  be  made  with  the  half-dried  meat 
instead  of  with  the  fresh.  Where  extreme  accuracy  is 
required  the  meat  is  to  be  dried  in  a  vacuum  over  sulphuric 
acid  instead  of  on  the  water-bath  (Pfliiger  and  Argutinsky). 
If  it  is  desired  to  make  the  nitrogen  determination  with  the 
fresh  meat,  weigh  it  off  on  a  piece  of  tin-foil,  fold  this,  place 
it  in  the  Kjeldahl  flask,  and  determine  the  nitrogen  in  the 
usual  manner. 

4.  Determination  of  Fat  or  Determination  of  the  Ether 
Extract.  This  is  best  made  with  the  fresh  meat.  According 
to  the  amount  of  fat  present,  weigh  off  accurately  an  aver- 
age sample  of  3  to  5  g.  in  a  large  weighing-glass,  pour  over  it 
about  30  cc.  of  absolute  alcohol,  stir  thoroughly  with  a  thin 
glass  rod,  which  is  then  washed  off  with  alcohol,  cork,  and  let 
stand  for  twenty-four  hours.  Filter  and  transfer  the  meat 
powder  completely  to  the  filter.  Evaporate  the  alcoholic 
extract  to  dryness  and  treat  the  residue  with  ether,  filter, 
wash  with  ether,  and  place  the  ether  extract,  after  concen- 
trating, in  the  Soxhlet  extraction-flask.  Place  the  filter  con- 
taining the  meat  powder  in  the  thimble  of  the  Soxhlet  appa- 
ratus. For  further  details  see  ' '  Determination  of  Fat  in  the 
Faeces,"  page  212. 

Pfliiger  and  Dormeyer  recommend,  instead  of  this  method, 
one  which  depends  on  the  dissolving  of  the  meat  by  digestion 
and  extraction  of  the  solution  obtained  with  ether  (Pfliiger's 


MEAT.  219 

Archiv,  65,  90,  1896).  This  method  gives  somewhat  higher 
results,  however,  and  there  is  more  chance  of  lactic  acid  being 
mixed  -^ith  the  fat  (or  f at  +  cholesterin  +  lecitliin)  in  this 
method  than  in  the  one  above  described. 

5.  Determination  of  the  Total  Phosphorus.  This  deter- 
mination is  made  in  the  same  way  as  the  corresponding 
determination  ^^^th  the  fseces,  1-1.5  g.  of  the  meat  powder 
being  fused  with  20  to  30  g.  -of  the  oxidizing  mixture  (using 
20  to  25  or  30  to  35  cc.  of  nitric  acid). 

6.  Determination  of  the  Total  Sulphur.  This  deter- 
mination may  be  made  with  tlie  fresh  meat  as  well  as  with 
the  dried  powder. 

(a)  Method  with  the  Fresh  Meat.  About  5  g.  of  meat 
are  accurately  weighed,  placed  in  a  long-necked  flask  (the 
residue  sticking  to  the  vessel  is  washed  in  with  nitric  acid), 
covered  with  nitric  acid  of  about  1.48  specific  gravity,  and 
heated  with,  this  on  the  water-bath  until  the  development  of 
red  fumes  has  completely  ceased.  Dilute  the  solution  with 
water,  place  it  in  a  porcelain  dish  (if  the  quantity  of  fat  is 
very  large,  the  solution  must  be  filtered  when  perfectly  cold 
and  the  filter  thoroughly  washed),  evaporate  on  the  water- 
bath  to  dryness,  dissolve  the  residue  in  5  to  6  g.  of  dry  sodium 
carbonate  (which  nmst  be  absolutely  free  from  sulphates) 
and  water,  place  in  a  platinum  dish,  add  3  g.  of  potassium 
nitrate,  evaporate  to  drjmess,  and  heat  slowly  till  fused. 
When  cold,  dissolve  the  perfectly  white  fused  mass  in  water, 
heat  the  solution  in  a  flask  (in  the  neck  of  which  a  funnel  is 
placed)  with  hydrochloric  acid  until  red  fumes  cease  to 
escape,  and  evaporate  in  a  porcelain  dish  on  the  water-bath 
to  complete  drj'ness.  Then  evaporate  twice  more  with 
hydrochloric  acid,  dissolve  in  water  (if  the  solution  is  not 
clear  it  must  be  filtered  from  the  silicic  acid  and  the  filter 
thoroughly  washed),  precipitate  the  hot  solution  with 
barium  chloride,  and  filter  after  twenty-four  hours,  etc. 


220  QUANTITATIVE  ANALYSIS. 

^233.46 parts BaS04  =  32.06  parts  sulphur.     See  the  "De- 
termination of  Sulphur  in  Urine,"  page  207. 

(b)  With  the  dry  meat  powder  the  determination  is  made 
with  about  1.5  g.  and  30  g.  of  the  oxidizing  mixture  in  the 
same  way  as  in  the  "  Determination  of  Sulphur  in  the  Urine/' 
see  page  207. 


V. 

ANALYSIS  OF  MILK. 

1.  Determination  of  the  Amount  of  Water.  Place  5  or 
10  cc.  of  milk  in  a  weighed  dish,  preferably  a  platinum  dish, 
evaporate  to  dryness  on  the  water-bath,  heat  to  constant 
weight  at  105°,  and  weigh. 

If  the  greatest  possible  accuracy  is  desired,  the  milk  must 
be  weighed,  not  measured,  and  the  drj^  residue  must  also  be 
protected  from  the  air  in  order  that  it  may  not  take  up  water 
during  the  weighing.  This  applies  to  all  similar  determina- 
tions. Both  these  objects  may  be  attained  by  using  a  plati- 
num crucible  for  the  determination.  This  is  placed  in  a 
large  weighing-glass,  the  glass  is  then  closed  and  the  weight 
of  the  whole  determined.  Five  to  ten  cubic  centimeters  of 
the  milk  are  then  introduced  into  the  crucible,  the  weighing- 
glass  is  closed,  and  the  quantity  of  the  milk  is  determin'ed  by 
weighing  again.  The  crucible  containing  the  dry'  residue  is 
also  weighed  in  the  weighing-glass. 

2.  Determination  of  the  Amount  of  Ash.  The  dry  resi- 
due is  carefully  carbonized,  then  more  strongly  heated,  but 
not  ignited  at  too  high  a  heat,  until  the  carbon  is  completely 
burned.  If  complete  combustion  cannot  be  attained  in  this 
way,  extract  the  half-burned  residue  by  cautiously  warming 
with  water,  filter  through  an  ashless  filter,  etc.,  as  described 
under  "Fa-ces"  and  "Meat." 

3.  Determination  of  Fat.      (a)  Let  5  to  10  cc.  of  milk 
drop  on  kaolin  or  burnt  gypsum  or  sand,  contained  in  the 

221 


222  QUANTITATIVE  ANALYSIS. 

paper  cartridge  of  the  Soxhlet  extraction  apparatus  (in  case  a 
closed  Schleicher  and  Schiill  extraction-thimble  is  not  availa- 
ble, this  cartridge  may  be  placed  in  a  cylinder  made  out  of  per- 
forated sheet  metal  and  closed  at  one  end),  dry  by  long  heat- 
ing at  105°,  and  extract  for  three  hours  in  the  Soxhlet  appara- 
tus with  anhydrous  ether. 

The  determination  of  the  dry  residue  may  also  be  com- 
bined with  the  determination  of  the  fat.  For  this  purpose 
dry  the  extraction-thimble  containing  the  kaolin  and  the 
milk  to  constant  weight.  Determine  the  loss  of  weight 
which  it  undergoes  when  extracted  with  ether.  This  must 
be  the  same  as  the  weight  of  the  fat.  Frequently  traces  of 
kaolin  or  gypsum  are  carried  over  mechanically  into  the 
ether  extract.^  In  this  case  the  ether  extract  must  of  course 
be  filtered  before  it  is  evaporated,  and  then,  too,  the  weight  of 
the  fat  will  not  coincide  with  the  loss  of  weight  of  the  extrac- 
tion-thimble. 

(h)  Warm  25  cc.  of  milk  with  the  same  amount  of  hydro- 
chloric acid  of  1.12  specific  gravity  for  some  time  in  a  flask 
on  the  water-bath,  let  cool,  and  transfer  to  a  separating-fun- 
nel,  rinsing  with  warm  water.  "Rinse  the  flask  several  times 
Avith  ether,  which  is  poured  into  the  separating-funnel  until 
the  volume  of  the  ether  equals  that  of  the  aqueous  fluid. 
Then  shake  with  the  ether,  separate  the  ether  extract,  and 
shake  once  or  twice  more  with  ether.  The  ether  extracts 
are  freed  from  hydrochloric  acid  by  shaking  with  water  and 
filtered  through  a  dry  filter  which  is  washed  mth  ether.  By 
evaporating  the  ether  the  fat  is  obtained.  This  frequently 
requires  to  be  purified  by  dissolving  once  more  in  ether. 

4.  Determination  of  the  Total  Nitrogen  according  to 
Kjeldahl.  Five  cubic  centimeters  of  milk  are  heated  in  a 
Kjeldahl  flask  with  20  cc.  of  concentrated   sulphuric   acid, 

1  When  a  paper  cartridge  made  by  Schleicher  and  Schiill  is  used  this 
is  less  liable  to  occur. 


MILK.  223 

0.5  g.  of  copper  sulphate,  and  10  g.  of  potassium  sulphate,  at 
first  gently  and  then  more  strongly,  until  the  contents  of 
the  flask  have  a  light-blue  or  green  color.  Place  25  cc.  of 
"fifth-normal  acid  in  the  receiver.  For  further  details  see  the 
''Determination  of  Nitrogen  according  to  Kjeldahl"  in  the 
chapter  on  ' '  TJrine." 

If  the  amount  of  proteid  be  calculated  by  multiplying  the 
amount  of  nitrogen  obtained  by  6.25,  as  is  customary,  the 
result  -will  be  somewhat  too  high.  According  to  I.  Munk, 
it  is  better  to  use  the  factor  6.0  for  cow's  milk,  and  for  hu- 
man milk  5.77.  See  the  "  Determination  of  Total  Proteid  " 
below. 

5.  Separate  Determination  of  Casein  and  Albumin  (accord- 
ing to  Schlossmann^).  Dilute  10  cc.  of  milk  with  3-5  parts 
of  water  and  cautiously  warm  to  40°  over  a  free  flame  or, 
better,  in  a  water-bath,  then  add  1  cc.  of  concentrated  solution 
of  potash-alum,  and  stir  thoroughly.  If  a  flocculent  precipi- 
tate, which  settles  quickly,  does  not  form,  then  continue  to 
add  the  alum  solution,  0.5  cc.  at  a  time,  until  the  coagulation 
and  precipitation  take  place  completely.  Of  course  time 
(half  a  minute)  must  be  allowed  before  each  addition  of 
the  alum  solution  for  the  settling  of  the  precipitate;  the 
temperature  is  to  be  kept  constant  at  40°.  A  slight  excess 
of  the  alum  solution  makes  no  difference.  After  the  com- 
pletion of  tjie  precipitation  let  stand  a  few  minutes  and  then 
filter.  When  the  filtrate  is  perfectly  clear,  which  may  require 
filtering  two  or  three  times  through  the  same  filter,  wash  the 
precipitate  a  few  times  on  the  filter  with  water  and  determine 
the  nitrogen  by  the  Kjeldahl  method.  From  the  amount 
of  nitrogen  found  calculate  the  amount  of  casein  by  nmlti- 
plying  by  6.37. 

Add  to  the  filtrate  10  cc.  of  tannin  solution,^  filter  off  the 

»  Z.eit.  f.  physiol.  Chernie,  22,  213. 

'  The  mixture  recommended  by  Almen,  consisting  of  4  g.  of  tannin,/ 


224  QUANTITATIVE  ANALYSIS. 

voluminous  precipitate  which  forms,  and,  after  washing- 
three  times  with  fresh  water,  determine  the  amount  of  nitro- 
gen by  the  Kjeldahl  method.  From  the  amount  of  nitrogen 
found  calculate  the  amount  of  albumin  and  globulin  by 
multiplying  by  6.37. 

6.  Determination  of  the  Total  Proteid  according  to  Ritt- 
hausen  and  I.  Munk.^  Place  10  cc.  of  human  or  cow's  milk 
in  a  250-cc.  beaker,  dilute  with  water  to  100  cc.  (with  human 
milk  dilution  to  60  cc.  is  sufficient),  heat,  and  add  1  to  2  cc. 
of  alum  solution,  then  when  the  fluid  just  begins  to  boil  2  to  5 
cc.  of  a  paste  of  cupric  hydroxide,  and  continue  the  boiling  for 
some  minutes.  The  finely  flocculent  precipitate,  which  set- 
tles quickly,  as  soon  as  the  mixture  has  been  coagulated  by 
heating,  is  filtered  while  still  warm,  washed  on  the  filter  with 
hot  water,  and  the  whole  filter  treated  while  still  moist  accord- 
ing to  Kjeldahl. 

The  cupric  hydroxide  is  prepared  according  to  Stutzer 
as  follows:  100  g.  of  crystallized  copper  sulphate  are  dis- 
solved in  5  liters  of  water  and  2.5  g.  of  glycerine  are  added. 
Dilute  sodium  hydroxide  solution  is  then  added  until  the  fluid 
reacts  alkaline,  the  cupric  hydroxide  is  then  filtered  off  and. 
ground  with  water  containing  5  g.  of  glycerine  to  the  liter. 
By  repeated  decanting  and  filtering  the  last  traces  of  alkali 
are  removed.  The  product  remaining  on  the  filter  is  then 
ground  and  diluted  with  water  which  contains  10  per  cent, 
of  glycerin,  so  that  it  forms  a  homogeneous  mass  which  may 
be  measured  out  with  a  pipette.  This  is  kept  in  the  dark  in  a 
well-closed  bottle.  The  amount  of  copper  oxide  in  the  pasty 
mass  may  be  determined  by  evaporating  a  measured  volume 


8  cc.  of  25  per  cent,  acetic  acid,  and  190  cc.  of  40  to  50  per  cent,  alcohol, 
gives  the  best  results. 

1  See  Ritthausen,  Jour.  f.  prakt.  Chemie  N.  F.  15,  329;  Emil  Pfeiffer,, 
Analyse  der  Milch.  Wiesbaden,  1887..  I.  Munk,  Virchow's  Arch.  134,, 
501  (1893). 


MILK.  225 

to  dnness  and  igniting  the  residue.  It  is  advisable  only  to 
prepare  small  quantities  at  a  time,  using  about  20  g.  of 
copper  sulphate. 

7.  Determination  of  Milk-sugar.  Dilute  the  filtrate  and 
wash-water  freed  from  the  albumin  (see  the  "  Determination 
of  Total  Proteid  according  to  Ritthausen  and  Munk  "  or  the 
method  of  Soxhlet  given  below)  to  a  definite  volume  (with 
20  cc.  of  milk  to  140  or  160  cc),  fill  a  burette  with,  this  solution, 
and  titrate  20  cc.  of  Fehluig's  solution +  80  cc.  of  water  with 
it.  (See  chapter  on  ''Urine.")  Twenty  cubic  centimeters 
of  Fehling's  solution  correspond  to  0.135  g.  of  anhydrous 
milk-sugar  (C12H22O11). 

Instead  of  titrating  we  may  add  to  40  cc,  of  Fehling's 
solution +  80  cc.  of  water,  heated  to  boiling,  30  cc.  of  the 
above  fluid,  continue  the  heating  for  six  to  seven  minutes, 
collect  the  precipitated  cuprous  oxide  on  a  weighed  filter 
and  weigh  as  such,  or  convert  it  into  copper  sulphide  or 
metallic  copper  and  weigh  this.  "We  may  also  remove  the 
casein  and  albumin  in  one  operation  according  to  Soxhlet. 
Twenty-five  cubic  centimeters  of  milk  are  mixed  with  400  cc. 
of  water,  a  few  drops  of  acetic  acid  are  added,  and  the  solu- 
tion heated  to  boihng.  When  cold  dilute  to  500  cc.  and  fil- 
ter through  a  dry  filter.  One  hundred  cubic  centimeters 
of  the  filtrate  =  5  cc.  of  milk  are  then  boiled  with  50  cc.  of 
Fehling's  solution  for  six  minutes,  etc.  Since  the  reduction 
equivalent  of  milk-sugar  for  copper  oxide  in  alkaline  solu- 
tion is  not  constant,  according  to  Soxlilet,  but  varies  ac- 
cording to  the  concentration  of  the  milk-sugar  solution,  an 
empirical  table  (see  pages  226  and  227)  nmst  be  used  for 
the  calculation.  This  has  been  established  by  Soxhlet  and 
calculated  (hrectly  for  milk-sugar  by  E.  Wein. 

8.  Determination  of  the  Total  Phosphorus.  Ten  cubic 
centimeters  of  milk  are  dropped  upon  30  g.  of  the  oxidizing 
mixture,  contained  in  a  platinum  dish,  evaporated  to  dry- 


226 


QUANTITATIVE  ANALYSIS. 

TABLE  TO  DETERMINE  THE  AMOUNT  OF 


<0 

ft 
a 

O 

o 

03 
M 

ft 
ft 
O 

o 

oj 

S3 

ft 

8- 
o 

oj 
bO 
3 

a 
% 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

100 

71.6 

135 

97.6 

170 

123.9 

205 

150.7 

101 

72.4 

136 

98.3 

171 

124.7 

206 

151.5 

102 

73.1 

137 

99.1 

172 

125.5 

207 

152.2 

103 

73.8 

138 

99.8 

173 

126.2 

208 

153.0 

104 

74.6 

139 

100.6 

174 

127.0 

209 

153.7 

105 

75.3 

140 

101.3 

175 

127.8 

210 

154.5 

106 

76.1 

141 

102.0 

176 

128.5 

211 

155.2 

107 

76.8 

142 

102.8 

177 

129.3 

212 

156.0 

108 

77.6 

143 

103.5 

178 

130.1 

213 

156.7 

109 

78.3 

144 

104.3 

179 

130.8 

214 

157.5 

110 

79.0 

145 

105.1 

180 

131.6 

215 

158.2 

111 

79.8 

146 

105.8 

181 

132.4 

216 

159.0 

112 

80.5 

147 

106.6 

182 

133.1 

217 

159.7 

113 

81.3 

148 

107.3 

183 

133.9 

218 

160.4 

114 

82.0 

149 

108.1 

184 

134.7 

219 

161.2 

115 

82.7 

150 

108.8 

185 

135.4 

220 

161.9 

116 

83.5 

151 

109.6 

186 

136.2 

221 

162.7 

117 

84.2 

152 

110.3 

187 

137.0 

222 

163.4 

118 

85.0 

153 

111.1 

188 

137.7 

223 

164.2 

119 

85.7 

154 

111.9 

189 

138.5 

224 

164.9 

120 

86.4 

155 

112.6 

190 

139.3 

225 

165.7 

121 

87.2 

156 

113.4 

191 

140.0 

226 

166.4 

122 

87.9 

157 

114.1 

192 

140.8 

227 

167.2 

123 

88.7 

158 

114.9 

193 

141.6 

228 

167.9 

124 

89.4 

159 

115.6 

194 

■  142.3 

229 

168.6 

125 

90.1 

160 

116.4 

195 

143.1 

230 

179.4 

126 

90.9 

161 

117.1 

196 

143.9 

231 

170.1 

127 

91.6 

162 

117.9 

197 

144.6 

232 

170.9 

128 

92.4 

163 

118.6 

198 

145.4 

233 

171.6 

129 

93.1 

164 

119.4 

199 

146.2 

234 

172.4 

130 

93.8 

165 

120.2 

200 

146.9 

235 

173.1 

131 

94.6 

166 

120.9 

201 

147.7 

236 

173.9 

132 

95.3 

167 

121.7 

202 

148.5 

237 

174.6 

133 

96.1 

168 

122.4 

203 

149.2 

238 

175.4 

134 

96.9 

169 

123.2 

204 

150.0 

239 

176.2 

MILK. 
BflLK-SUGAR  FROM  THE  AA^IGHT  OF  COPPER. 


227 


u 

<a 

D. 

1 

OS 

to 

3 
1 

u 

(U 

ft 
ft 

a 

i 

be 

3 

ft 

a 

a 

c 

oj 

bc 
3 

u 

Hi 

a 
ft 

& 

u 

OS 

f 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

mg. 

240 

176.9 

275 

204.3 

310 

232.2 

345 

259.8 

241 

177.7 

276 

205.1 

311 

232.9 

346 

260.6 

242 

178.5 

277 

205.9 

312 

233.7 

347 

261.4 

243 

179.3 

278 

206.7 

313 

234.5 

348 

262.3 

244 

180.1 

1  279 

207  .5 

314 

235.3 

349 

263.1 

245 

180.8 

280 

208.3 

315 

236.1 

350 

263.9 

246 

181.6 

281 

209.1 

316 

236.8 

351 

264.7 

247 

182.4 

282 

209.9 

317 

237.6 

352 

265.5 

248 

183.2 

2S3 

210.7 

318 

238.4 

353 

266.3 

249 

184.0 

284 

211.5 

319 

239.2 

354 

267.2 

250 

184.8 

285 

212.3 

320 

240.0 

355 

268.0 

251 

185.5 

286 

213.1 

321 

240.7 

356 

268.8 

252 

186.3 

287 

213.9 

322 

241.5 

357 

269.6 

253 

187.1 

288 

214.7 

323 

242.3 

358 

270.4 

254 

187.9 

289 

215.5 

324 

243.1 

359 

271.2 

255 

188.7 

290 

216.3 

325 

243.9 

360 

272.1 

256 

189.4 

291 

217.1 

326 

244.6 

361 

272.9 

257 

190.2 

292 

217.9 

327 

245.4 

362 

273.7 

258 

191.0 

293 

218.7 

328 

246.2 

363 

274.5 

259 

191.8 

294 

219.5 

329 

247.0 

364 

275.3 

260 

192.5 

295 

220.3 

330 

247.7 

365 

276.2 

261 

193.3 

296 

221.1 

331 

248.5 

366 

277.1 

262 

191.4 

297 

221.9 

332 

429.2 

367 

277.9 

263 

194.9 

298 

222.7 

333 

250.0 

368 

278.8 

264 

195.7 

299 

223.5 

334 

250.8 

369 

279.6 

265 

196.4 

300 

224.4 

335 

251.6 

370 

280.5 

266 

197.2 

301 

225.2 

336 

252.5 

371 

281.4 

267 

198.0 

302 

225.9 

337 

253.3 

372 

282.2 

268 

198.8 

303 

226.7 

338 

254.1 

373 

283 . 1 

269 

199.5 

304 

227.5 

339 

254.9 

374 

283.9  . 

270 

200.3 

305 

228.3 

340 

255.7 

375 

284.8 

271 

201.1 

306 

229.1 

341 

256.5 

376 

285.7 

272 

201.9 

307 

229.8 

342 

257.4 

377 

286.5 

273 

202.7 

308 

230.6 

343 

258.2 

378 

287.4 

274 

203.5 

309 

231.4 

344 

259.0 

379 

288.2 

228  QUANTITATIVE  ANALYSIS. 

ness,  and  fused. ^  Then  proceed  as  directed  under  "Faeces/^ 
page  214,  using  30  to  35  cc.  of  nitric  acid.  Instead  of  this 
method  we  may  also  use  sulphuric  acid  and  ammonium  nitrate 
for  the  oxidation  according  to  the  method  of  A.  Neumann 
(see  page  215). 

9.  Determination  of  the  Total  Sulphur.  Ten  cubic  cen- 
timeters of  milk  are  evaporated  to  dryness  with  30  g.  of  the 
oxidizing  mixture  and  fused.  Then  proceed  as  directed 
under  ' '  Urine,"  page  207. 

^  By  working  very  carefully  the  oxidizing  mixture  may  be  heated  ta 
fusion  directly  after  adding  the  milk. 


VI. 

ANALYSIS  OF  BREAD,  ETC. 

For  the  analysis  of  white  bread  it  is  best  to  take  a  whole 
roll,  weigh  It,  cut  it  up  over  a  sheet  of  paper  into  slices  about 
a  centimeter  thick,  place  them  without  loss  in  an  evaporating- 
dish,  weigh  agam  as  a  check,  heat  on  the  water-bath  or  in  an 
air-bath  until  the  pieces  are  dry  enough  to  be  powdered   let 
cool,  weigh,  grind,  and  place  the  powder  in  a  tight  glass- 
stoppered  bottle.     This  material  is  used  for  the  analysis 
If  the  volume  of  a  single  roll  is  too  large  for  this  method  of 
procedure,  a  mixture  of  the  crust  and  crumb  is  prepared 
which  corresponds  as  nearly  as  possible  to  the  proportion  of 
crust  and  crumb  of  the  bread,  and  with  this  we  proceed  as 
directed  above. 

For  the  determination  of  the  amount  of  water  and  ash 
take  2  to  3  g.  of  the  powder;  for  the  nitrogen  determination 
about  1.5  to  1.8  g.,  placing  25  to  30  cc.  of  fifth-normal  acid 
in  the  receiver  (the  digestion  must  be  conducted  very  care- 
fully at  first);  for  the  determination  of  fat,  extract  about  4  g 
in  a  Soxhlet  apparatus,  or,  better,  boil  the  same  quantity 
with  75  to  100  cc.  of  dilute  hydrochloric  acid  (1:2)  till  dis- 
solved and  then  extract  with  ether;  the  dry  residue  obtained 
by  evaporating  the  ether  extract  must  be  again  dissolved  in 
ether  to  purify  it.     For  the  determination  of  the  carbohy- 
drates, heat  2  to  3  g.  (of  course  all  these  quantities  arc  to  be 
accurately  weighed),  according  to  the  method  of  Marker 
with  lactic  acid  solution,  etc.     (See  the  "Determination  of 

229 


230  QUANTITATIVE  ANALYSIS. 

the  Carbohydrates  in  the  Fseces,"  page  213.)  If  the  sugar 
is  to  be  determined  gravimetrically,  take  50  cc.  of  the  Feh- 
hng's  solution,  100  cc.  of  water,  and  25  cc.  of  the  solution  ulti- 
mately obtained  from  the  bread.  For  further  details  of  the 
methods  consult  the  earlier  chapters. 

According  to  L.  Liebermann  ^  this  method  gives  too  low- 
results  on  account  of  the  destruction  of  sugar  by  the  long- 
continued  heating.  He  recommends  the  following  method: 
About  10  g.  of  the  substance  are  boiled  for  one  and  one-haK 
hours  on  a  sand-bath  with  100  cc.  of  2  per  cent,  hydrochloric 
acid  in  a  250-300-cc.  flask  connected  with  a  reflux  condenser. 
Then  the  fluid  is  almost  neutralized  with  sodium  hydroxide 
solution,  filtered  into  a  liter  measuring-flask,  rinsing  and 
washing  with  hot  water.  Dilute  to  one  liter  and  take  out  20 
cc.  for  the  sugar  determination  -v^ith  FehUng's  solution. 
According  to  L.  Liebermann  there  is  no  danger  of  the  2  per 
cent,  hydrochloric  acid  converting  the  cellulose  into  sugar. 
Instead  of  filtering  and  washing,  the  fluid,  together  with  sus- 
pended material,  may  be  diluted  to  one  liter  and  then  filtered 
through  a  dry  filter. 

Liebermann  uses  a  peculiar  method  for  the  determina- 
tion of  the  cuprous  oxide.  The  precipitated  oxide  is  filtered 
off,  washed,  dissolved  in  hydrochloric  acid,  and  the  solution 
reduced  in  a  weighed  platinum  dish  with  a  small  piece  of 
zinc.  The  liquid  is  poured  off  from  the  precipitated  copper, 
which  is  then  washed  a  few  times  with  water,  alcohol,  and 
ether,  dried  at  100°  and  weighed. 

For  the  phosphorus  determination  fuse  1.5  to  1.8  g.  with 
30  g.  of  the  oxidizing  mixture  (30-35  cc.  of  nitric  acid). 

1  Maly's  Jahrb.  f .  Thierchemie,  1886,  55. 


VII. 

ANALYSIS  OF  BLOOD. 

If  the  blood  has  stood  for  any  length  of  time,  it  must  be 
thoroughly  shaken  in  order  to  avoid  errors  which  may  arise 
from  the  precipitation  of  the  blood-corpuscles.  Small  quan- 
tities of  blood  cannot  be  easily  measured  without  rinsing  out 
the  pipette,  which  is  not  permissible  with  aqueous  solutions, 
and  also  causes  a  small  error  in  the  case  of  blood.  Instead 
of  this  we  may  weigh  off  the  necessary  quantities,  but  in 
this  case  we  must  refer  the_  results  to  1  kg.  of  blood  (instead 
of  one  liter).  It  is  also  very  advantageous  to  dilute  the 
blood  beforehand.  Let  25  cc.  of  the  blood  flow  into  a  100- 
cc.  measuring-flask,  rinse  the  pipette  with  distilled  water, 
and  fill  up  to  the  100-cc.  mark.  Take  out  portions,  mixing 
thoroughly  by  shaking  each  time  before  any  of  the  blood  is 
removed. 

1.  For  the  determination  of  the  water  and  ash  5  cc.  of 
the  blood  or  10  to  20  cc.  of  the  diluted  blood  is  sufficient. 

2.  For  the  nitrogen  determination  heat  5  cc.  of  the  blood 
with  10  to  15  cc.  of  sulphuric  acid  and  0.5  g.  of  CuSO^  (placing 
60  cc.  ■of  fifth-normal  acid  in  the  receiver),  or  use  10  cc.  of  the 
diluted  blood  (with  35  cc.  of  the  fifth-normal  acid  in  the 
receiver),  and  titrate  back  with  tenth-normal  ammonia  solu- 
tion. 

3.  For  the  phosphorus  determination  heat  5  cc.  of  blood 

or  20  cc.  of  the  diluted  blood  with  30  g.  of  the  oxidizing  mix- 

231 


232  QUANTITATIVE  ANALYSIS. 

ture  (30-35  cc.  of  nitric  acid),  or  oxidize  according  to  the 
method  of  A.  Neumann,  page  215. 

4.  For  the  sulphur  determination  fuse  the  same  quanti- 
ties with  the  oxidizing  mixture,  or  oxidize  first  with  nitric 
acid,  as  in  the  case  of  meat. 

5.  For  the  determination  of  fat  20  to  25  cc.  of  the  blood 
are  weighed  off  (less  may  also  be  used),  poured  into  five 
times  its  volume  of  absolute  alcohol  contained  in  a  wide- 
necked  glass-stoppered  bottle,  and  repeatedly  shaken.  (The 
pipette  must  not  be  rinsed  here.  It  is  best  to  fasten  it  in  a 
clamp  and  let  it  stand  for  some  time,  when  the  blood  gradu- 
ally collects  and  may  be  blown  out.  If  the  blood  has  been 
weighed  [in  a  large  weighing-glass],  then  rinse  out  the  glass 
with  alcohol.)  Filter  next  day  through  a  dry  filter,  wash 
once  with  absolute  alcohol,  and  let  the  filter  dry.  Evaporate 
the  alcoholic  solution  to  dryness,  dissolve  the  residue  in 
ether,  place  the  ether  extract  in  the  Soxhlet  flask  and  the 
filter  with  its  contents  (or  the  coagulum  alone,  if  it  can  be 
separated  from  the  filter  without  any  loss)  in  the  extraction- 
thimble,  and  determine  the  fat  in  the  usual  manner. 

6.  Determination  of  Iron.  Evaporate  to  dryness  on 
the  water-bath  between  15  and  20  g.  (or  cc.)  of  blood  in  a 
platinum  or  porcelain  dish  and  cautiously  carbonize  until 
vapors  cease  to  be  evolved.  The  residue  is  then  warmed 
with  dilute  hydrochloric  acid  free  from  iron,  the  solution 
filtered  through  a  filter  containing  no  iron,  and  the  filter 
washed  with  water  until  the  filtrate  no  longer  has  an  acid 
reaction.  The  filter  with  the  carbon  is  dried,  and  the  dish 
also.  The  filter  is  then  placed  in  the  dish  and  ignited  until 
the  carbon  is  completely  burned.  The  hydrochloric  acid 
solution  is  then  poured  into  the  dish,  about  twenty  drops  of 
dilute  sulphuric  acid  are  added,  the  contents  of  the  dish  are 
evaporated  to  complete  dryness  on  the  water-bath  and 
ignited.     Cover  the  residue  remaining  after  ignition  with  a 


BLOOD.  233 

mixture  of  three  volumes  of  concentrated  sulphuric  acid 
.and  two  volumes  of  water,  warm  until  dissolved,  and  dilute 
to  50  to  100  cc.  To  determine  the  iron  in  the  solution  we 
make  use  of  the  reaction  of  potassium  permanganate  on  a 
solution  of  ferrous  sulphate  containing  free  sulphuric  acid. 
This  takes  place  according  to  the  following  equation : 

lOFeSO^  +  2KMnO,  +  9H2SO4  = 

Ferrous  Potassium         Sulphuric 

sulphate      permanganate  acid 

SFe^CSOJa  +  2MnS0,  +  2HKSO4  +  SH^O. 

Ferric  Manganous     Monopotassium 

sulphate  sulphate  sulphate 

In  the  solution  obtained  above,  however,  the  iron  is  pres- 
ent as  a  ferric  salt.  If  we  wish  to  determine  the  iron  by 
titration  with  potassium  permanganate,  we  must  first  con- 
vert it  into  a  ferrous  salt.  This  may  be  most  readily  accom- 
plished by  means  of  some  metallic  zinc  (weighed  quantity 
about  1  g.).  The  reduction  is  made  in  an  atmosphere  of 
carbon  dioxide  and  is  continued  until  the  fluid  has  become 
perfectly  colorless  and  the  zinc  has  completely  dissolved 
(some  dilute  sulphuric  acid  must  eventually  be  added).  Let 
the  contents  of  the  flask  cool  completely  in  the  atmosphere 
of  carbon  dioxide,  and  titrate  with  a  solution  of  potassium 
permanganate  whose  strength  has  been  accurately  deter- 
mined. To  prepare  this  solution  weigh  off  accurately  0.32 
g.  of  pure  potassium  permanganate,  dissolve  in  water,  and 
dilute  to  the  volume  of  one  liter.  This  solution  may  be 
standardized  by  means  of  either  oxalic  acid  or  ferrous  ammo- 
nium sulphate. 

Standardization  with  Oxalic  Acid.  Weigh  off  accu- 
rately 0.63  g.  of  perfectly  pure  oxalic  acid  which  has  not 
■effloresced,  dissolve  in  water,  and  dilute  to  one  liter.  Add 
to  25  cc.  of  this  solution  a  few  cubic  centimeters  of  dilute 
sulphuric  acid,  heat  in  a  flask  on  the  wire  gauze  to  boiling, 
.and  let  the  permanganate  solution  run  in  from  a  burette 


234  QUANTITATIVE  ANALYSIS. 

until  the  color  of  the  permanganate  no  longer  completely 
disappears  on  shaking.  The  first  permanent  faint  red  color 
indicates  the  end-point  of  the  reaction.  This  is  best  seen 
when  the  flask  is  placed  on  a  sheet  of  white  paper.  If  exactly 
25  cc.  are  used,  then  the  strength  of  the  solution  is  correct 
and  each  cubic  centimeter  of  the  permanganate  solution  cor- 
responds to  0.56  mg.  of  iron.  If  such  an  exact  agreement  is 
not  found,  it  is  best  not  to  change  the  strength  of  the  solu- 
tion, but  to  calculate  its  value.  For  example,  if,  instead  of 
25  cc,  25.6  cc.  were  used,  then  the  solution  is  too  dilute  and 

0  56x25 
1  cc.  does  not  correspond  to  0.56  mg.  of  iron,  but  to  -^rr^-^i — 

25.6 

=  0.5468  mg. 

Standardization  with  Ferrous  Ammonium  Sulphate.  Weigh 
off  exactly  3.924  g.  of  pure,  dry  ferrous  ammonium  sulphate, 
reS04,(NHj2S04+6H20  (none  of  the  crystals  should  be 
yellow-colored),  dissolve  in  water  which  has  previously  been 
boiled,  and  dilute  the  solution  with  boiled  and  cold  water  to 
one  liter.  Titrate  as  with  the  oxalic  acid,  but  at  room  tem- 
perature. 

Titration  of  the  Solution  Obtained  from  the  Blood.  The 
titration  with  the  permanganate  solution  is  carried  out 
exactly  in  the  same  manner  as  in  standardizing  with  the 
ferrous  ammonium  sulphate  solution.  Since,  however,  the 
zinc  contains  traces  of  iron,  a  check  experiment  is  made  with 
the  zinc  alone.  This  procedure  does  not  take  into  account 
the  slight  reducing  action  of  the  carbon  contained  in  the 
zinc.^  The  amount  of  metallic  iron  in  oxyhsemoglobin  is 
approximately  0.4  per  cent.  The  amount  of  the  oxyhse- 
moglobin is  therefore   obtained  from  the  amount  of  iron. 

found  by  multiplying  by  rr-r  =  250. 

^  To  avoid  this  action  it  is  also  recommended  to  dilute  the  solution, 
obtained  exactly  to  100  cc.,let  settle,  and  then  take  out  with  the  pipette 
50  cc.  for  the  determination. 


BLOOD.  235 

Simplification  of  the  Method  of  Preparing  the  Ash.     The 

above  method  of  preparmg  the  ash  may  be  simphfied  by- 
extracting  the  carbon  with  water  instead  of  with  hydro- 
cliloric  acid.  The  aqueous  extract  may  be  disregarded,  as 
it  contains  no  iron/  and  the  ash  remaining  may  be  directly 
dissolved  in  the  sulphuric  acid  of  the  concentration  given 
above.  The  complete  oxidation  of  the  ash  takes  place  with 
somewhat  more  difficulty. 

The  preparation  of  the  ash  may  also  be  accomplished 
more  conveniently  by  not  evaporating  the  blood  directly 
and  then  igniting,  but  by  treating  it  first  according  to  Kjel- 
dahl.  Fifteen  to  twenty  cubic  centimeters  of  blood  are 
heated  with  20  to  30  cc.  of  sulphuric  acid  and  some  mercuric 
oxide  or  mercuric  chloride  solution.  Ultimately  some  more 
hot  sulphuric  acid  is  added  and  the  heating  is  continued  until 
the  solution  has  become  colorless.  When  cold  this  is  placed 
in  a  platinum  dish,  the  flask  rinsed  with  a  small  quantity  of 
water,  the  excess  of  sulphuric  acid  driven  off  on  the  sand- 
bath,  and  then  ignited  completely.  Although  this  method 
may  perhaps  appear  more  roundabout,  yet  it  is  really  more 
convenient.  The  method  of  previously  heating  with  sul- 
phuric acid  in  a  Kjeldahl  flask  may  also  be  advantageously 
used  for  the  quantitative  determination  of  many  other  ash 
constituents,  as  wefl  as  for  the  detection  of  metallic  poisons. 

The  destructiori  of  the  organic  matter  may  be  more 
readily  accomplished  according  to  the  method  of  A.  Neumann 
by  using  with  the  sulphuric  acid  the  same  amount  of  ammo- 
nium nitrate. 

Gravimetric  Determination  of  the  Iron.  If  only  single 
determinations  of  iron  are  to  be  made,  instead  of  a  long 
series,  the  gravimetric  determination  as  ferric  phosphate, 
FePO^,  is  more  convenient.     For  this  purpose  add  to  the 


*  It  may,  however,  contain  iron  if  the  extract  is  highly  colored. 


236  QUANTITATIVE  ANALYSIS. 

solution  obtained  from  the  ash  and  containing  all  of  the  iron 
as  ferric  salt  a  few  cubic  centimeters  of  sodium  phosphate 
solution,  make  alkaline  with  ammonia,  and  acidify  with 
acetic  acid.  The  precipitate  of  ferric  phosphate  resulting  is 
collected  on  an  ash-free  filter,  washed,  dried,  ignited  (in  a 
porcelain  crucible),  and  weighed.  One  hundred  parts  corre- 
spond to  52.98  parts  of  FejOg  or  to  37.09  parts  of  Fe. 

Since  by  this  method  it  makes  no  difference  if  the  solution 
still  contains  traces  of  organic  matter,  the  evaporation  of 
the  diluted  sulphuric  acid,  ignition,  and  dissolving  again  in 
sulphuric  acid  (see  above)  may  be  omitted.  (In  the  titra- 
tion with  permanganate  this  procedure  is  also  frequently 
unnecessary).  Frequently  also  all  the  iron  passes  so  com- 
pletely into  the  hydrochloric  acid  solution  (especially  in  the 
determination  of  iron  in  the  organs,  which  is  made  in  an 
exactly  analogous  manner)  that  it  is  sufficient  to  treat  the 
ash  remaining  with  a  little  hydrochloric  acid,  filter  the 
diluted  solution,  add  it  to  the  first  hydrochloric  acid  extract, 
and  then  precipitate  the  iron  directly  from  the  hydrochloric 
acid  solution.  Hydrochloric  acid  solutions  cannot,  however, 
be  titrated  with  potassium  permanganate  solution. 


VIII. 

DETERMINATION  OF  HYDROCHLORIC  ACID  IN  THE 
GASTRIC  JUICE  ACCORDING  TO  SJOQVIST. 

Twenty-five  cubic  centimeters  of  the  hydrochloric  acid 
solution  A  (see  page  33)  and  the  same  amount  of  the 
lactic  acid  solution  are  mixed  in  a  dry  beaker.  Ten  cubic 
centimeters  of  this  mixture  are  then  measured  off  with 
a  pipette,  placed  in  an  evaporating-dish,  a  few  drops  of 
albumose  solution  and  a  small  quantity  of  absolutely  pure 
barium  carbonate  ^  added,  and  the  mixture  heated  on  the 
water-bath.  During  the  heating  the  mixture  is  thoroughly 
stirred  with  a  thin  glass  rod.  This  is  then  rinsed  off  and  the 
mixture  evaporated  to  dryness.  The  dry  residue  now  con- 
sists of  barium  chloride,  barium  lactate,  excess  of  barium 
carbonate,  albumose,  and  the  salts  which  may  be  present 
and  which  are  never  absent  from  the  fluids  of  the  stomach. 
This  is  now  heated  over  a  free  flame  and  ignited  gently  until 
most  of  the  carbon  is  consumed  (complete  oxidation  is  super- 
fluous). By  this  means  the  organic  substances  are  burnt  up 
or  carbonized,  the  barium  lactate  is  converted  into  barium 
carbonate,  while  the  barium  chloride  remains  unchanged. 
Let  the  dish  cool,  extract  the  residue  with  hot  water,  and 
filter  through  a  small  filter  of  thin  paper  (for  example, 
Schleicher  and  Schiill's  No.  590),  of  at  most  9  cm.  diameter. 

•  If  the  barium  carbonate  contains  alkali  carbonate,  which  is  very 
frequently  the  case,  the  amount  of  hydrochloric  acid  found  will  be  too 

low. 

237 


238  QUANTITATIVE  ANALYSIS. 

Only  barium  chloride,  together  with  some  salts  present  in 
solution,  passes  through  the  filter,  while  the  insoluble  barium 
carbonate  remains  behind.  Wash  the  dish  with  hot  water, 
pour  into  the  filter  which  was  previously  used,  and  wash 
until  all  the  barium  chloride  has  been  completely  washed  out 
of  the  filter.  By  working  carefully  this  may  be  accom- 
plished without  increasing  the  volume  of  the  filtrate  and  the 
wash- water  to  more  than  50  to  60  cc.  We  may  also  con- 
tinue the  washing  somewhat  longer  and  then  cautiously 
evaporate  to  50  cc.  In  any  case  the  last  wash- water  which 
is  thought  to  be  free  from  barium  chloride  is  collected  by 
itself  and  tested  with  silver  nitrate  and  nitric  acid.^  The 
filtrate  and  the  wash-water  contain  all  the  hydrochloric 
acid  of  the  gastric  juice  combined  with  barium.  The  amount 
of  barium  is  hence  a  direct  measure  of  the  amount  of  hydro- 
chloric acid. 

To  determine  the  amouni  of  barium,  acidify  the  filtrate 
and  wash-water  with  a  few  drops  of  hydrochloric  acid,  heat 
in  a  beaker  on  the  wire  gauze  until  boiling  begins,  add  about 
4  to  5  cc.  of  dilute  sulphuric  acid  (also  previously  heated), 
then  heat  further  on  the  water-bath  until  the  barium  sul- 
phate settles,  leaving  a  clear  solution.  Filter  through  a 
close,  ash-free  filter  ^  of  about  9  cm.  diameter,  transfer  the 
precipitate  completely  to  the  filter — the  filtrate  must  be 
perfectly  clear;  if  it  is  not,  it  must  be  again  passed  through 
the  same  filter — and  wash  until  a  portion  of  the  wash-water 
is  no  longer  rendered  turbid  by  silver  nitrate  solution  or  by 
barium   chloride   solution.     Then   fill   the   filter   once   with 

1  The  original  direction,  to  wash  until  a  portion  of  the  wash-water  is 
no  longer  made  turbid  with  sulphuric  acid,  is  not  to  be  adhered  to,  since 
such  a  point,  in  consequence  of  the  slight  solubility  of  the  barium  car- 
bonate in  water,  is  not  to  be  attained.  Too  long-continued  washing  may 
therefore  also  lead  to  a  plus  error. 

'  Suitable  filter-papers  are  the  No.  590  of  Schleicher  and  Schiill,  and 
the  ash-free  baryta  filter-papers  Nos.  400  and  412  of  Dreverhoff  in  Dresden. 


DETERMINATION  OF  HYDROCHLORIC  ACID.         239 

absolute  alsohol  and  once  with  ether,  let  the  excess  of  ether 
evaporate,  place  the  filter  together  with  the  barium  sulphate 
m  a  weighed  platinum  crucible,  heat,  at  first  gently  and  then 
more  strongly,  with  the  crucible  half  open,  until  all  the  car- 
bon is  burned,  and  weigh  when  cold  (see  page  206).  One 
molecule  of  barium  sulphate,  BaSO^,  corresponds  to  two 
molecules  of  hydrochloric  acid,  HCl;  233.46  parts  by  weight 
correspond  to  72.92  parts  of  hydrochloric  acid,  HCl.  The 
entire  determination  should  be  made  in  duplicate  as  a  check. 
Instead  of  precipitating  the  barium  as  sulphate  and 
weighing  we  may  also  proceed  as  follows :  Add  to  the  aqueous 
solution  of  barium  chloride  obtained  ammonia  and  ammonium 
carbonate,  filter  off  the  precipitated  barium  carbonate,  wash 
thoroughly,  and  dissolve  in  dilute  hydrochloric  acid.  For 
this  purpose  it  is  best  to  wash  the  barium  carbonate  into  a 
beaker,  dissolve  it  in  dilute  hydrochloric  acid,  filter  the  solu- 
tion through  the  filter  which  was  used  to  collect  the  barium 
carbonate,  and  wash.  The  solution  is  evaporated  to  com- 
plete dryness  on  the  water-bath  to  remove  any  hydrochloric 
acid  still  present,  a  few  cubic  centimeters  of  water  are  added, 
and  the  solution  is  again  evaporated  to  dryness.  The  residue 
is  dissolved  in  water  and  titrated  with  a  dilute  solution  of 
silver  nitrate  of  known  strength,  after  the  addition  of  suffi- 
cient potassium  chromate  solution.^  A  solution  of  silver 
nitrate  containing  2.9054  g.  of  AgNOg  to  the  liter,  1  cc.  of 
which  equals  0.001  g.  NaCl,  should  be  used.  If  this  silver 
solution  is  used  the  percentage  amount  of  HCl  in  the  gastric 

nX 3.646    . 
juice  may  be  calculated  from  the  fornmla  x=      ^^     ,  m 

which  n  indicates  the  number  of  cubic  centimeters  of  the  sil- 
ver solution  used  for  10  cc.  of  the  gastric  juice. 


'  The  amount  of  the  potassium  chromate  solution  added  must  not  be 
too  small,  as  the  potassium  chromate  reacts  with  the  barium  chloride  to 
form  insoluble  barium  chromate  and  potassium  chloride. 


IX. 

QUANTITATIVE  DIGESTION  EXPERIMENTS. 

For  a  short  series  of  experiments  fresh  fibrin  and  coag- 
lated  egg-albumen  may  be  used;  for  a  longer  series  only- 
material  containing  a  fixed  amount  of  water  can  be  used. 
For  example,  fibrin  which  has  been  treated  with  alcohol 
and  ether  and  then  powdered,  coagulated  egg -albumen 
treated  with  alcohol  and  ether,  etc.  In  any  case,  care  must 
be  taken  that,  in  any  series  of  experiments,  the  same  material 
is  always  used.  This  should  be  prepared  beforehand  in 
large  quantities,  and  it  must  be  kept  in  closed  vessels,  since 
the  amount  of  water  which  it  contains  must  not  change. 
Blood-serum  which  has  been  preserved  by  the  addition  of 
chloroform  may  also  be  used.^  The  chloroform  must  be 
expelled  by  means  of  an  air-current  before  using  the  serum. 
The  results  are  not  quite  equivalent,  but  depend  upon  the 
nature  of  the  material  used.  SHght  disturbing  influences 
do  not  often  appear  when  fresh  fibrin  is  used,  but  only  when 
hard-boiled  egg-albumen  is  used.  Furthermore,  weak  dis- 
turbing influences  are  sometimes  more  perceptible  when 
pepsin-hydrochloric  acid  is  used  than  when  the  extract  of 
the  lining  of  the  stomach  is  made  use  of.  Finally,  the  dura- 
tion of  the  digestion  is  also  of  influence.     It  is  often  neces- 

^  Fluid  egg-albumen  can  no  longer  be  used,  since  we  have  learned  that 
it  contains  ovomucoid,  which  necessarily  causes  an  error  when  we  base  our 
opinion  regarding  the  digestibility  on  the  quantity  of  the  portion  dis- 
solved during  the  digestion. 

240 


QUANTITATIVE  DIGESTION  EXPERIMENTS.         241 

sar}^,  in  order  to  recognize  disturbing  influences,  to  shorten 
the  duration  of  the  digestion  even  to  four  hours.  The 
answer  to  the  question  whether  a  substance  disturbs  the 
digestion  cannot,  therefore,  strictly  speaking,  be  given  in 
general,  but  only  applies  for  the  special  conditions  adhered 
to  in  the  experiment.  Of  course  there  are  also  substances 
which  interfere  with  the  digestion  even  when  the  conditions 
for  the  digestion  are  the  most  favorable,  e.g.,  larger  quanti- 
ties of  sugar,  gum,  or  plant-mucus.^  ^^^len  fibrin  or  coagu- 
lated albumin,  etc.,  is  used  we  may  either  determine  the 
undissolved  albumin  or  the  dissolved  or  both.  When  an 
albumin  solution  is  used  the  determination  of  that  which 
remains  undissolved  is  of  course  omitted.  If  we  Umit  our- 
selves to  the  determination  of  the  portion  remaining  undis- 
solved, then  the  precipitate  formed  on  neutralization  would 
be  included  among  the  products  of  the  digestion,  which  is 
scarcely  justifiable.  At  all  events,  it  is  preferable  to  deter- 
mme  the  undissolved  albumin  +  the  precipitable  albumin, 
on  the  one  hand  and  the  peptonized  (album  ose  and  peptone) 
on  the  other,  or  to  determine  the  quantity  of  the  albumin 
present  at  the  beginning  of  the  digestion  and  the  quantity 
of  the  peptonized  material  in  the  mixture  of  digestion  prod- 
ucts.^ It  is  advisable  to  replace  the  very  long  and  also  not 
quite  accurate  determination  of  the  dry  residue  by  the 
Kjeldahl  nitrogen  determination.  One  example  of  this  kind 
of  experiment  may  suffice. 

Blood -serum  is  treated  with  an  equal  amount  of  water, 
shaken  thoroughly,  and  exactly  neutralized  with  dilute 
hydrochloric  acid.  

»Mugdan,  Berl.  klin.  Wochenschr. ,  1891,  No.  32. 

'  If  we  also  estimate  at  the  same  time  the  quantity  of  the  coagulable 
albumin  (including  the  residue  remaining  undissolved)  in  the  mixture  of 
the  digestion  products,  then  we  have  a  check  on  the  correctness  of  the 
analysis:  the  sum  of  these  values  and  the  albumoses  must  equal  the  quan- 
tity of  the  albumin  used. 


242  QUANTITATIVE  ANALYSIS.  ' 

Place  in  each  of  a  series  of  flasks  or  bottles  (with  stop- 
pers) 50  cc.  of  pepsin-hydrochloric  acid.  In  each  of  another 
series  place  50  cc.  of  the  pepsin-hj^drochloric  acid  which 
contains  the  weighed  quantity  of  the  material  whose  influ- 
ence is  to  be  tested,  or  add  to  a  number  of  portions  of  the 
pepsin-hydrochloric  acid  the  material  to  be  tested  and  dis- 
solve it  by  shaking  without  warming.  Then  place  in  each 
flask  20  cc.  of  the  albmnin  solution,  shake  thoroughly,  and 
digest  it  at  40°,  shaking  repeatedly  during  the  digestion. 
To  avoid  accidental  errors,  each  mixture  must  be  prepared 
in  duplicate. 

To  determine  the  amount  of  nitrogen  in  the  albumin 
solution,  heat  20  cc.  of  the  solution  with  15  cc.  of  concen- 
trated sulphuric  acid,  10  g.  of  potassium  sulphate,  and  0.5  g. 
of  copper  sulphate.  This  determination  is  also  to  be  made 
in  duplicate.  The  heating  must  be  done  at  first  with  great 
care,  otherwise  the  determination  may  be  lost  from  foam- 
ing. When  the  heating  has  continued  about  one  and  one- 
half  hours  and  the  oxidation  is  not  then  completed,  it  is 
advisable  to  let  cool,  add  10  to  15  cc.  of  sulphuric  acid  and 
heat  again.  When  the  oxidation  is  completed,  which  may 
be  accomplished  even  without  the  addition  of  potassium 
permanganate,  let  cool,  dilute  the  solution,  let  cool  again, 
put  it  in  a  measuring-flask  and  fill  up  to  100  cc.  Twenty- 
five  or  fifty  cubic  centimeters  of  the  well-mixed  solution  are 
used  for  the  determination  of  the  ammonia  or  amount  of 
nitrogen.  To  collect  the  ammonia  20  to  40  cc.  of  fifth-normal 
hydrochloric  acid  are  sufficient.  The  amount  of  albumin 
is  obtained  by  multiplying  the  amount  of  nitrogen  found  by 
6.25. 

After  the  digestion  of  the  albumin  has  continued  the  desired 
number  of  hours,  neutralize  the  mixture  with  dilute  caustic 
soda  solution  (half-  or  fourth-normal),  heat  to  boiling,  add 
acetic  acid  to  faintly  acid  reaction  and  5  cc.  of  concentrated 


QUANTITATIVE  DIGESTION  EXPERIMENTS  243 

sodium  chloride  solution  in  order  to  completely  precipitate  all 
the  albumin.  Then  let  cool,  place  the  entire  fluid  together  with 
the  precipitate  in  a  100-  or  200-cc.  measuring-flask,  rinse  the 
vessel  thorouglily,  fill  up  to  the  mark,  filter  through  a  dry 
filter,  and  take  a  quarter  or  half  of  the  whole  amount  for  the 
nitrogen  determination. 

The  amomit  of  nitrogen  contained  in  the  pepsin-hydro- 
chloric acid  is  so  small  that  it  may  be  neglected.  It  must  be 
considered,  however,  when  an  extract  of  the  mucus  membrane 
of  the  stomach  is  used.  Of  course  the  amount  of  nitrogen 
in  the  substance  tested  is  also  to  be  taken  into  consideration. 

It  is  not  always  possible  to  proceed  as  above ;  quite  often 
the  method  must  be  modified.  For  the  arrangement  of 
these  experiments  see  Virchow's  Arch.  120,  353;  122,  238; 
127,  514;  Berl.  klin.  Wochenschr.  1891,  No.  32;  Virchow's 
Arch.  150,  260  (1897). 


X. 

QUANTITATIVE  DETERMINATION  OF  GLYCOGEN.* 

Place  100  g.  of  the  ground  meat  or  organ  and  100  cc.  of 
60  per  cent,  potassium  hydroxide  solution  in  a  200-cc.  flask. 
Use  the  purest  potassium  hydroxide  made  by  "Merck." 
The  strength  of  the  potassium  hydroxide  solution  is  deter- 
mined accurately  with  standard  hydrochloric  acid. 

Before  placing  the  flask  in  the  boiling  water-bath  shake 
it  once  or  twice  in  order  to  mix  the  contents  thoroughly,  and 
repeat  the  shaking  after  the  flask  has  been  heated  one- 
quarter  to  one-half  of  an  ho'ur.  As  soon  as  the  liquid  has 
assumed  the  temperature  of  the  boiling  water-bath  and  no 
further  expansion  takes  place,  close  the  flask  with  a  rubber 
stopper. 

After  heating  for  two  hours,  take  the  flask  out  of  the  boil- 
ing water-bath,  empty  it  into  a  400-cc.  measuring-flask,  and 
rinse  it  out  with  boiling  water.  When  the  liquid  is  cold  fill 
the  400-cc.  flask  up  to  the  mark  with  sterilized  water  and 
filter  the  solution  through  glass  wool  until  the  filtrate  is  quite 
clear  or  only  slightly  opalescent.  From  a  burette  measure 
off  100  cc.  of  the  filtered  meat  solution  into  a  300-cc.  beaker, 
add  100  cc.  of  96  per  cent.  (Tralles)  alcohol,  and  mix  thor- 
oughly with  a  glass  rod.  The  precipitate  of  glycogen  settles 
very  rapidly,  so  that  it  may  be  filtered  off  even  after  a  quarter 
of  an  hour.     It  is  safer,  however,  to  wait   some   hours  or, 

'  Pfliiger  in  Pfliiger's  Arch.  93,  163. 

244 


QUANTITATIVE  DETERMINATION  OF  GLYCOGEN.      245 

best,  overnight.  Filter  through  a  15-cm.  filter  (Munktell)  and 
wash  the  precipitate  with  a  mixture  of  one  volume  of  15  per 
cent,  potassium  hydroxide  solution  and  two  volumes  of  alco- 
hol (96%  Tr.).  Collect  this  wash-fluid  in  the  beaker  in 
which  the  glycogen  was  precipitated  and  pour  it  through  the 
filter  twice.  Then  wash  the  glycogen  on  the  filter  with  90 
per  cent,  alcohol. 

After  the  alcohol  has  drained  thoroughly  draw  a  well- 
cleaned  rubber  tube  over  the  end  of  the  funnel-tube  and 
close  it  with  a  pinch-cock.  Under  the  rubber  tube  place  the 
empty  beaker  in  which  the  precipitation  of  glycogen  took 
place.  It  makes  no  difference  if  a  little  glycogen  sticks  to  the 
walls  of  the  beaker.  Then  fill  the  funnel  full  of  sterilized  but 
cold  water.  After  one-half  to  one  hour  the  glycogen  will  have 
almost  completely  dissolved.  Open  the  pinch-cock  and  let 
the  solution  run  into  the  beaker.  Then  close  the  rubber 
tube  again,  pour  some  more  water  into  the  funnel,  wait  till 
all  the  glycogen  has  dissolved,  and  again  open  the  pinch-cock. 
After  the  water  has  run  out  a  second  time  close  the  pinch- 
cock  again,  fill  the  filter  half  full  of  water,  and  wash  the  fine 
green  dust  from  the  paper  with  the  aid  of  a  fine  brush.  This 
dust  is  insoluble  in  water.  Finally  allow  the  wash-water  to 
flow  into  the  beaker.  Then  place  a  small  piece  of  litmus 
paper  in  the  filtrate  and  let  hydrochloric  acid,  1.19  specific 
gravity,  run  in  from  a  burette,  drop  by  drop,  mixing  thor- 
oughly with  a  glass  rod  until  the  alkali  is  exactly  neutralized.' 

Then  place  a  i^mnel  in  a  500-cc.  measuring-flask  and 
pour  in  the  neutralized  glycogen  solution  with  all  the  neces- 
sary precautions  observed  in  quantitative  analysis. 

Measure  out  of  the  burette  25  cc.  of  hydrochloric  acid 
of  1.19  specific  gravity  into  the  beaker  and  pour  this  also  into 
the  500-cc.  flask.  Then  place  the  flask  with  its  funnel  under 
the  exit-tube  of  the  funnel  which  contains  the  glycogen 
filter.    Fill  the  beaker  with  water  and  pour  it  on  the  filter 


246  QUANTITATIVE  ANALYSIS. 

in  order  to  wash  out  the  last  traces  of  the  glycogen,  and  add 
it  to  the  500-cc.  flask.  Repeat  this  rinsing  until  the  500-cc. 
flask  is  almost  but  not  quite  filled  to  the  mark.  Finally 
allow  a  few  cubic  centimeters  of  the  fluid  to  run  into  a  test- 
tube  and  add  several  cubic  centimeters  of  96  per  cent,  alco- 
hol. No  turbidity  should  result.  Then  close  the  500-cc. 
flask  with  a  rubber  stopper  and  shake  the  mixture  thoroughly. 
It  contains  approximately  2.2  per  cent,  hydrochloric  acid. 
These  directions  must  be  modified : 

1.  When  such  a  small  quantity  of  glycogen  is  present  that 
an  exact  analysis  cannot  be  carried  out.  In  this  case  we 
precipitate  the  glycogen  with  an  equal  volume  of  alcohol, 
not  from  100  cc.  of  the  filtered  meat  solution,  but  from  200  or 
300  cc,  and  proceed  as  directed  above. 

2.  When  the  glycogen  is  to  be  estimated  in  a  very  small 
organ,  e.g.,  in  the  liver  of  a  chicken.  In  this  case  weigh  off 
10  g.  of  the  finely  chopped  liver  and  put  it  into  a  small  flask 
with  10  cc.  of  60  per  cent,  potash  solution.  After  heating 
for  two  hours  in  the  water-bath,  cool  and  fill  up  to  40  cc. 
with  water.  Filter  through  glass  wool  and  use  25  to  30  cc. 
for  precipitation  with  an  equal  volume  of  96  per  cent.  alcohoL 
Filter  through  a  small  Swedish  filter  and  proceed  as  directed 
above. 

The  solution  of  the  glycogen  on  the  filter  must  be  made 
so  that  not  more  than  100  cc.  of  the  filtrate  with  2.2  per  cent. 
of  hydrochloric  acid  is  obtained  in  a  100-cc.  flask. 

Determination  of  the  Glycogen  by  Conversion  into  Glucose. 

The  flask  containing  the  glycogen  solution  is  placed  in  a. 
boiling-water-bath  and  closed  with  a  rubber  cork  as  soon  as 
the  expansion  of  the  liquid  caused  by  the  heat  has  ceased. 
After  heating  for  three  hours,  remove  the  flask  from  the 
bath,  let  it  cool,  and  fiU  up  to  the  mark  with  water.  Then 
filter  the  sugar  solution  through  a  dry  Swedish  filter  into  a. 


QUANTITATIVE  DETERMINATION  OF  GLYCOGEN.    247 

500-cc.  flask.  The  glucose  in  this  solution  is  then  determined 
gravimetrically   (see   Determination  of  Glucose,  page  200). 

According  to  Salkowski  ^  it  is  much  better  not  to  treat 
the  fresh  liver  with  caustic  potash  solution,  but  to  subject 
it  to  a  preliminary  treatment  mth  alcohol  and  ether.  This 
is  done  by  extracting  the  finely  chopped  liver  with  absolute 
alcohol  and  then  with  ether,  thus  converting  it  into  a  fine 
powder.  This  dissolves  comparatively  readily  in  2-3  per 
cent,  potassium  hydroxide  solution,  the  rapidity  of  the  solu- 
tion depending  upon  the  completeness  of  the  pulverization 
of  the  liver.  If  this  has  been  well  done,  then  the  solution  in 
the  caustic  potash  takes  place  in  a  few  minutes.  The  solu- 
tion is  brown-colored  and  not  quite  clear  omng  to  the  pres- 
ence of  undissolved  calcium  phosphate.  Only  compara- 
tively small  quantities  of  the  liver  powder,  5  to  10  g.,  need 
to  be  taken  for  the  glycogen  determination. 

The  alkaline  solution  is  clarified  by  letting  it  stand — it  is 
advisable  to  take  only  an  aliquot  part,  in  order  to  avoid 
filtering,  about  four-fifths — and  then  it  is  precipitated  with 
double  its  volume  of  alcohol,  the  precipitate  washed,  first  with 
66  per  cent,  alcohol,  then  with  stronger  alcohol,  and  dried  at 
105°.  The  glycogen  thus  obtained  contains  only  a  trace  of 
nitrogen,  though  it  leaves  considerable  ash  when  ignited. 
It  may  be  determined  as  glucose  after  hydrolyzing  according 
to  Pfliiger's  method  given  above. 

The  liver  powder,  prepared  as  stated  above,  also  dis- 
solves very  quickly  in  artificial  gastric  juice.  With  5  g.  of 
the  powder  Salkowski  found  no  glycogen  in  the  residue  left 
after  digesting  for  forty-six  hours,  nor  did  the  coagulum 
resulting  from  boiling  the  solution,  after  it  had  been  nearly 
neutralized,  contain  even  a  trace  of  glycogen.  From  the 
filtrate,  after  evaporating  it  to  about  150  cc,  the  addition 

'  Zeit.  f.  physiologische  Chemie,  36,  257. 


248  QUANTITATIVE  ANALYSIS. 

of  double  the  volume  of  alcohol  precipitated  glycogen,  con- 
taining far  less  ash  than  that  which  was  obtained  from  the 
alkaline  solution.  This  glycogen  contained  only  traces  of 
nitrogen,  and  its  solution  gave  no  precipitate  with  Briicke's 
reagent  and  hydrochloric  acid. 

A  simple  method  depending  on  the  auto-digestion  of  the 
liver  is  described  by  Austin,  Virchow's  Arch.  150,  185  (1897). 
It  yields  approximately  correct  results. 


APPENDIX  I. 

REAGENTS. 

The  reagents  mentioned  in  the  text,   unless  otherwise 
stated,  are  solutions  of  the  following  concentration  or  of  sub- 
stances which  must  show  the  following  standard  of  purity : 
Acetic  Acid,  containing  30  per  cent,  of  acetic  acid. 
Alcohol.    ]\Iust  be  colorless  and  leave  no  residue  on  evapora- 
tion. 
Alkaline  Barium  Chloride  Solution.    Two  volumes  of  baryta- 
water  and  one  volume  of  barium  chloride  solution. 
Almen's  Solution.     Dissolve  4  g.  of  tannin  in  8  cc.  of  25  per 
cent,  acetic  acid  and  then  add  190  cc.  of  40  to  50  per 
cent,  alcohol. 
Ammonia.    A  solution  of  ammonia  in  water,  specific  gravity 

0.96,  containing  about  10  per  cent.  NHg. 
Ammonium  Carbonate.     One  part  ^  of  the  commercial  ammo- 
nium carbonate,  one  part  of  ammonia,  and  four  parts  of 
water.     IMust  stand  some  days  before  being  used. 
Ammonium  Chloride,  1:10.* 

Ammonium  Molybdate  in  acid  solution.  Dissolve  50  g.  of 
molybdic  acid  in  200  g.  of  8-10  per  cent,  ammonia  and 
pour  the  solution  into  750  g.  of  nitric  acid,  specific 
gravity  1.2.  Let  stand  for  some  days  in  a  warm  place 
and  then  decant. 
Ammonium  Oxalate,  1 :  25. 

'  By  parts  is  always  meant  parts  by  weight. 

*  Distilled  water  is  always  to  be  used  as  the  solvent;  1 ;  10  means  1  part 
by  weight  of  ammonium  chloride  dissolved  in  10  parts  by  weight  of  water. 
The  water,  of  course,  is  to  be  measured. 

249 


250  QUANTITATIVE  ANALYSIS. 

Barium  Carbonate,  precipitated  from  barium  chloride  solu- 
tion by  ammonium  carbonate  and  well  washed. 

Barium  Chloride,  1:10. 

Barium  Nitrate,  1 :  12. 

Baryta-water.  One  part  of  crystallized  caustic  baryta  heated 
with  fifteen  parts  of  water  and,  after  cooling,  filtered. 

Basic  Lead  Acetate.  Lead  subacetate  solution,  commercial. 
(Liquor  Plumbi  Acetici  Ph.  G.  IV.) 

Bromine-water.  Water  shaken  with  an  excess  of  bromine 
and  allowed  to  stand  till  the  bromine  has  settled. 

Briicke's  Reagent  (potassium  mercuric  iodide  solution). 
Heat  a  solution  of  potassium  iodide  containing  100  g.  of 
KI  in  the  liter,  and  add  mercuric  iodide  as  long  as  it  dis- 
solves. After  cooling  decant  from  the  red  crystals  and 
add  a  few  crystals  of  potassium  iodide. 

Calcium  Chloride.     Dry  pure  calcium  chloride,  1 :  10. 

Cochineal  Tincture.  Five  grams  of  cochineal,  150  cc.  of 
alcohol,  and  100  cc.  of  water  are  allowed  to  stand  for 
some  days  at  room  temperature;  the  solution  is  then 
poured  off  and  filtered.  The  cochineal  remaining  may 
be  used  over  again. 

Copper  Sulphate,  1 :  10. 

Ether.  Must  leave,  on  evaporation,  no  residue  having  an 
odor  or  an  acid  reaction. 

Ferric  Chloride,  3 :  100. 

Hydrochloric  Acid,  specific  gravity  1.183. 

Lead  Acetate,  1 :  10. 

Magnesia  Mixture.  One  part  of  crystallized  magnesium  sul- 
phate, two  parts  of  ammonium  chloride,  four  parts  of 
ammonia,  and  eight  of  water,  or  110  g.  of  magnesium 
chloride  (chemically  pure),  140  g.  of  ammonium  chloride, 
700  cc.  of  8  per  cent,  ammonia,  and  1300  parts  of  water 
(or  250  cc.  of  ammonia,  specific  gravity  0.91,  and  1750  cc. 
of  water).    Let  stand  for  some  days  before  using. 


APPENDIX  I.  251 

Magnesium  Sulphate,  cold,  saturated  solution. 

Mercuric  Chloride,  1 :  20. 

Millon's  Reagent.  Warm  one  part  of  mercury  with  two 
parts  of  nitric  acid,  specific  gravity  1.4,  until  the  mer- 
cury is  completely  dissolved.  Dilute  one  volume  of 
the  solution  with  two  volumes  of  water. 

0.  Nasse  (Arch.  f.  d.  ges.  Physiol.  83,  361  (1901) )  rec- 
ommends an  aqueous  solution  of  mercuric  acetate,  to 
which,  just  before  using,  a  few  drops  of  a  1  per  cent,  po- 
tassium nitrite  solution  are  to  be  added  and  also,  in  case 
the  reaction  is  not  distinctly  acid,  a  little  dilute  acetic  acid. 

Nessler's  Reagent.  Dissolve  50  g.  of  potassium  iodide  in  the 
same  quantity  of  water,  then  heat  and  add  a  hot,  concen- 
trated mercuric  cliloride  solution  until  some  mercuric 
iodide  remains  undissolved.  (Twenty  to  twenty-five 
grams  of  HgClg  are  required.)  Filter  and  add  150  g.  of 
potassium  hydroxide  dissolved  in  300  g.  of  water,  then 
dilute  to  one  liter,  add  5  cc,  more  of  the  mercuric  chlo- 
ride solution,  let  the  precipitate  settle  and  decant.  The 
solution  is  kept  in  small  closed  bottles.  It  must  be 
completely  saturated  with  mercuric  iodide,  otherwise 
the  reagent  is  not  very  sensitive. 

Nitric  Acid.  Must  be  free  from  hydrochloric  acid  and  color- 
less, and  free  from  nitrous  acid,  specific  gravity  1.2. 

Nylander's  Solution.  One  hundred  grams  of  caustic  soda 
solution  of  1.119  specific  gravity  (10.33  g.  NaOH),  4  g.  of 
potassium  sodium  tartrate,  and  2  g.  of  bismuth  subnitrate. 

Oxidizing  Mixture.  Three  parts  of  KNO3  and  one  part  of 
NajCOg,  dry  and  pure. 

Phosphotungstic  Acid,  1 :  20,  acidified  with  hydrochloric  acid. 

Platinum  Chloride.  Must  dissolve  and  give  a  clear  solution 
in  alcohol,  1 :10. 

Potassium  Chromate.  Yellow  chromate  of  potassium,  K2Cr04, 
1:20. 


•252  QUANTITATIVE  ANALYSIS. 

Potassium  Ferrocyanide,  1 :  10. 

Potassium  Hydroxide.  One  part  of  potassium  hydroxide 
(pure,  fused)  to  two  parts  of  water. 

Potassium  Nitrate.    Must  be  free  from  chlorides  and  sulphates. 

Potassium  Sulphocyanate,  1 :20. 

Rosolic  Acid,  one  part  in  100  to  200  parts  of  alcohol. 

Silver  Nitrate,  1 :  50. 

Sodium  Carbonate.  Dehydrated  and  a  saturated  solution, 
must  be  free  from  chlorides  and  sulphates. 

Sodium  Chloride.  Cold  saturated  solution  (water  shaken  for 
some  time  with  an  excess  of  powdered  sodium  chloride). 
One  hundred  cubic  centimeters  of  the  solution  contain 
31.84  g.  of  sodium  chloride. 

Sodium  Hydroxide,  containing  about  15  g.  of  sodium  hydrox- 
ide in  100  g.  of  the  solution,  specific  gravity  1.17. 

Sodium  Phosphate,  NazHPOi,  1 :  10. 

Sulphuric  Acid.  By  this  term  is  always  meant,  unless  other- 
wise stated,  an  acid  containing  200  g.  of  concentrated 
sulphuric  acid  in  the  liter. 

Tartaric  Acid,  pulverized. 

Turmeric-paper.  Digest  powdered  turmeric-root  with  alcohol, 
dip  bibulous  paper  in  the  solution,  and  dry  in  the  air. 

Zinc  Chloride  Solution,  alcohoHc.  Thick,  sirupy,  aqueous 
solution  diluted  with  alcohol  until  the  specific  gravity 
is  1.2. 


APPENDIX  II. 

TABLES  OF  THE  SPECIFIC  GRAVITIES  OF 
SOME    SOLUTIONS. 


1.  CAUSTIC  SODA, 


100  Grams 

1  Liter 

Specific 

Gravity 

at  15°. 

100  Grams 

1  Liter 

Specific 
Gravity 

contain 
NaOH  in 

contains 
NaOH  in 

contain 
NaOH  in 

contains 
NaOH  in 

at  15°. 

Grams. 

Grams. 

Grams. 

Grams. 

1.007 

0.61 

6 

1.190 

16.77 

200 

1.014 

1.20 

12 

1.200 

17.67 

212 

1.022 

2.00 

21 

1.210 

18.58 

225 

1.029 

2.71 

28 

1.220 

19.58 

239 

1.036 

3.35 

35 

1.231 

20.59 

253 

1.045 

4.00 

42 

1.241 

21.42 

266 

1.052 

4.64 

49 

1.252 

22.64 

283 

1.060 

5.29 

56 

1.263 

23.67 

299 

1.067 

5.87 

63 

1.274 

24.81 

316 

1.075 

6.55 

70 

1.285 

25.80 

332 

1.083 

7.31 

79 

1.297 

26.83 

348 

1.091 

8.00 

87 

1.308 

27.80 

364 

1.100 

8.68 

95 

1.320 

28.83 

381 

1.108 

9.42 

104 

1.332 

29.93 

399 

1.116 

10.06 

112 

1.345 

31.22 

420 

1.125 

10.97 

123 

1.357 

32.47 

441 

1.134 

11.84 

134 

1.370 

33.69 

462 

1.142 

12.64 

144 

1.383 

34.96 

483 

1.152 

13.55 

156 

1.397 

36.25 

506 

1.162 

14.37 

167 

1.410 

37.47 

528 

1.171 

15.13 

177 

1.424 

38.80 

553 

I.ISO 

15.91 

188 

1.438 

39.99 

575 

253 


254  TABLES  OF  THE  SPECIFIC  GRAVITIES. 

2.  CAUSTIC  POTASH. 


Specific 
Gravity 
at  15°. 

100  Grams 

1  Liter 

Specific 
Gravity 
at  15°. 

100  Grams 

1  Liter 

contain 
KOHin 

contains 
KOHin 

contain 
KOHin 

contains 
KOHin 

Grams. 

Grams. 

Grams. 

Grams. 

1.007 

0.9 

9 

1.190 

21.4 

255 

1.014 

1.7 

17 

1.200 

22.4 

269 

1.022 

2.6 

26 

1.210 

23.3 

282 

1.029 

3.5 

36 

1.220 

24.2 

295 

1.037 

4.5 

46 

1.231 

25.1 

309 

1.045 

5.6 

58 

1.241 

26.1 

324 

1.052 

6.4 

67 

1.252 

27.0 

338 

1.060 

7.4 

78 

1.263 

28.0 

353 

1.067 

8.2 

88 

1.274 

28.9 

368 

1.075 

9.2 

99 

1.297 

30.7 

398 

1.083 

10.1 

109 

1.320 

32.7 

432 

1.091 

10.9 

119 

1.345 

34.9 

469 

1.100 

12.0 

132 

1.370 

36.9 

506 

1.108 

12.9 

143 

1.397 

38.9 

543 

1.116 

13.8 

153 

1.424 

40.9 

582 

1.125 

14.8 

167 

1.453 

43.4 

631 

1.134 

15.7 

178 

1.483 

45.8 

679 

1.142 

16.5 

188 

1.530 

49.4 

756 

1.152 

17.6 

203 

1.580 

53.2 

840 

1.162 

18.6 

216 

1.615 

55.9 

902 

1.171 

19.5 

228 

1.634 

57.5 

940 

1.180 

20.5 

242 

3.  AMMONIA. 


Specific 
Gravity 
at  15°. 

Per  Cent. 
NH3. 

1  Liter 
contains 
NHsin 
Grams. 

Specific 
Gravity 
at  15°. 

Per  Cent. 
NH3. 

1  Liter 

contains 

NHsin 

Grams. 

0.970 

7.31 

70.9 

0.942 

15.04 

141.7 

0.968 

7.82 

75.7 

0.940 

15.63 

146.9 

0.966 

8.33 

80.5 

0.938 

16.22 

152.1 

0.964 

8.84 

85.2 

0.936 

16.82 

157.4 

0.962 

9.35 

89.9 

0.934 

17.42 

162.7 

0.960 

9.91 

95.1 

0.932 

18.03 

168.1 

0.958 

10.47 

100.3 

0.930 

18.64 

173.4 

0.956 

11.03 

105.4 

0.928 

19.25 

178.6 

0.954 

11.60 

110.7 

0.926 

19.87 

184.2 

0.952 

12.17 

115.9 

0.920 

21.75 

200.1 

0.950 

12.74 

121.0 

0.910 

24.99 

227.4 

0.948 

13.31 

126.2 

0.906 

26.31 

238.3 

0.946 

13.88 

131.3 

0.902 

27.65 

249.4 

0.944 

14.46 

136.5 

0.900 

28.33 

255 

APPENDIX  II. 


255 


4.  HYDROCHLORIC  ACID. 


Specific 

100  Grams 

1  Liter 

Specific 

100  Grams 

1  Liter 

Gravity 

contain 

contains 

Gravity 

contain 

contains 

jl5° 

HClin 

HCl  in 

^15° 

HCl  in 

HCl  in 

d^- 

Grams. 

Kilograms. 

d^- 

Grams. 

Kilograms. 

1.200 

39.11 

0.469 

1.115 

22.86 

0.255 

1.195 

38.16 

0.456 

1.110 

21.92 

0.243 

1.190 

37.23 

0.443 

1.105 

20.97 

0.232 

1.185 

36.31 

0.430 

1.100 

20.01 

0.220 

1.180 

35.39 

0.418 

1.095 

19.06 

0.209 

1.175 

34.42 

0.404 

1.090 

18.11 

0.197 

1.171 

33.65 

0.394 

i        1.085 

17.13 

0.186 

1.170 

33.46 

0.392 

1.080 

16.15 

0.174 

1.165 

32.49 

0.379 

1.075 

15.16 

0.163 

1.163 

32.10 

0.373 

1.070 

14.17 

0.152 

1.160 

31.52 

0.366 

1.065 

13.19 

0.141 

1.155 

30.55 

0.353 

1.060 

12.19 

0.129 

1.152 

29.95 

0.345 

1.055 

11.18 

0.118 

1.150 

29.57 

0.340 

1.050 

10.17 

0.107 

1.145 

28.61 

0.328 

1.045 

9.16 

0.096 

1.1425 

28.14 

0.322 

1.040 

8.16 

0.085 

1.140 

27.66 

0.315 

1.035 

7.15 

0.074 

1.135 

26.70 

0.303 

1.030 

6.15 

0.064 

1.130 

25.75 

0.291 

1.025 

5.15 

0.053 

1.125 

24.78 

0.278 

1.020 

4.13 

0.042 

1.120 

23.82 

0.267 

1.015 

3.12 

0.032 

5.  NITRIC  ACID. 


Specific 

100  Grams 

1  Liter 

Specific 

100  Grams 

1  Liter 

Gravity 

contam 

contains 

Gravity 

contain 

contains 

15° 

HNOsin 

HNO3  in 

wl5° 

HNOain 

HNOain 

4°' 

Grams. 

Kilograms. 

d^- 

Grams. 

Kilograms. 

1.520 

99.67 

1.515 

1.360 

57.57 

0.783 

1.509 

97. &i 

1.476 

1.345 

54.93 

0.739 

1.495 

91.60 

1.369 

1.335 

53.22 

0.710 

1.485 

87.70 

1.302 

1.315 

49.89 

0.656 

1.475 

84.45 

1.246 

1.295 

46.72 

0.605 

1.465 

81.42 

1.193 

1.275 

43.64 

0.556 

1.4.55 

78.60 

1.144 

1.250 

39.82 

0.498 

1.440 

74.68 

1.075 

1.225 

36.03 

0.441 

1.430 

72.17 

1.032 

1.195 

31.62 

0.378 

1.420 

69.80 

0.991 

1.185 

30.13 

0.357 

1.405 

66.40 

0.933 

1.175 

28.63 

0.336 

1.395 

64.25 

0.896 

1 .  165 

27.12 

0.316 

1.375 

60.30 

0.829 

1.155 

25.60 

0.296 

256  TABLES  OF  THE  SPECIFIC  GRAVITIES. 

6.  ALCOHOL.     60°F.  =  15M°C. 


15.56° 
15.56°" 

Volume, 

Weight 

15.56° 

Volume, 

Weight 

Per  Cent. 

Per  Cent. 

15.56°" 

Per  Cent. 

Per  Cent. 

0.89499 

68 

60.48 

0.84961 

85 

79.58 

0.89256 

69 

- 61.53 

0.84660 

86 

80.80 

0.89010 

70 

62.59 

0.84355 

87 

82.03 

0.88762 

71 

63.66 

0.84044 

88 

83.28 

0.88511 

72 

64.74 

0.83726 

89 

84.54 

0.88257 

73 

65.83 

0.83400 

90 

85.82 

0.88000 

74 

66.92 

0.83065 

91 

87.12 

0.87740 

75 

68.02 

0.82721 

92 

88.44 

0.87477 

76 

69.13 

0.82365 

93 

89.79 

0.87211 

77 

70.26 

0.81997 

94 

91.16 

0.86943 

78 

71.39 

0.81616 

95 

92.56 

0.86670 

79 

72.53 

0.81217 

96 

93.99 

0.86395 

80 

73.68 

0.80800 

97 

95.45 

0.86116 

81 

74.84 

0.80359 

98 

96.95 

0.85833 

82 

76.00 

0.79891 

99 

98.51 

0.85574 

83 

77.18 

0.79391 

100 

100.13 

0.85256 

84 

78.37 

INTERNATIONAL   ATOMIC  WEIGHTS. 


0=  16 

Aluminium Al         27. 1 

Antimony Sb  120.2 

Argon A  39.9 

Arsenic As         75 

Barium Ba  137.4 

Beryllium Be  9.1 

Bismuth Bi  208.5 

Boron B  11 

Bromine Br         79.96 

Cadmium Cd  112.4 

Caesium Cs  133 

Calcium Ca         40.1 

Carbon C  12 

Cerium Ce  140 

Chlorine CI  85.45 

Chromium Cr         52.1 

Cobalt Co         59 

Copper Cu         63.6 

Erbium Er  166 

Fluorine F  19 

Gadolinium Gd  156 

Gallium Ga         70 

Germanium Ge         73 . 5 

Gold Au  197.2 

Helium He  4 

Hydrogen H  1.008 

Indium In  114 

Iodine I  126.85 

Iridium Ir  193 

Iron Fe         55.9 

Krypton Kr         81.8 

Lanthanum La  138.9 

Lead Pb  206.9 

Lithium Li  7.03 

Magnesium Mg        24.36 

Manganese Mn        55 

Mercury Hg  200 

Mo'  ybdenum Mo  96 

Ne«xlymium Nd  143.6 


0  =  16 

Neon Ne        20 

Nickel Ni         58.7 

Niobium Nb        94 

Nitrogen N  14.04 

Osmium Os  191 

Oxygen... O  16 

Palladium Pd  106.5 

Phosphorus P  31 

Platinum Pt  194  8 

Potassium K  39.15 

Praseodymium Pr  140 . 5 

Radium Ra  225 

Rhodium Rh  103 

Rubidium Rb  85.4 

Ruthenium Ru  101.7 

Samarium Sa  150 

Scandium Sc  44.1 

Selenium Se  79 . 2 

Silicon Si  28.4 

Silver Ag  107.93 

Sodium Na  23.05 

Strontium Sr  87.6 

Sulphur S  32.06 

Tantalum Ta  183 

Tellurium Te  127.6 

Terbium Tb  160 

Thallium Tl  204.1 

Thorium Th  232.5 

Thulium Tu  171 

Tin Sn  119 

Titanium Ti  48.1 

Tungsten W  184 

Uranium U  238.5 

Vanadium V  51.2 

Xenon X  128 

Ytterbium Yb  173 

Yttrium Y  89 

Zinc Zn  65.4 

Zirconium Zr  90.6 

257 


INDEX. 


Absorption  spectra,  colored  plate  of, 

262. 
Acetoacetic  acid,  120. 
Acetone,  120. 
Achroodextrin,  74. 
Acid  albumin,  61. 
Adanikiewicz's  reaction,  5. 
Adenine,  135. 
Adipose  tissue,  141. 
Albumen  of  the  egg,  155. 
Albumin,  coagulated,  4. 

detection  in  urine,  114. 

estimation  in  urine,  195. 

estimation  in  milk,  223. 

putrefaction  of,  159. 

reactions,  62,  155. 

removal  from  urine,  183. 
Albumoses,  detection  in  urine,  115. 

reactions,  44 

separation  from  peptone,  42. 
Alkali  albuminate,  61. 
Alloxuric  bases,  24. 
Aluminiimi,  estimation  in  alum,  178. 
Ammonia,  detection  in  urine,  125. 

estimation  in  urine,  193. 

standard  solution  of,  IS 6. 
Ash,  determination  in  blood,  231. 

determination  in  bread,  229. 

determination  in  faeces,  211. 

determination  in  meat,  217. 

determination  in  milk,  221. 


Benzoic  acid,  105. 
Bile,  S5. 

acids,  86. 

crystallized,  85. 

mucin,  88. 

pigments,  detection  in  urine,  124. 


Biliary  calculi,  90. 
BiUrubin,  93. 
Biliverdin,  94. 
Bismuth  test  for  sugar,  12. 
Biuret  reaction,  45,  98. 
Blood,  analysis  of,  231. 

coagulation  by  heating,  57. 

conduct  towards  hydrogen  perox- 
ide, 49. 

corpuscles,  solution  of,  50. 

pigments,      detection     in      urine, 
122. 

reaction  with  gum  guaiacum,  49. 

spectroscopic  examination,  51. 

testing  the  reaction  of,  48. 
Blood-fibrin,  58. 
Blood-serum,  58. 
Bone,  137. 

mineral,  constituents  of,  140. 
Bread,  analysis  of,  229. 
Bromide  of  potassium,  detection  in 

urine,  126. 
Butter-fat,  10. 

Calcium,  estimation,  177. 

phosphate,  7. 
Calculi,  biliary,  90. 

urinar>',  128. 
Carbohydrates,  estimation  in  bread, 
229. 

estimation  in  ffeces,  213. 
Carbon  monoxide  haemoglobin.  52. 
CamifTerin,  32. 
Caseui,  8. 

estimation  in  mUk,  223. 
Cheese,  16. 
Chlorides,  detection  in  urine,  124. 

estimation  in  urine,  205. 

259 


260 


INDEX. 


Chlorine,  estimation  in  sodium  chlo- 
ride, 178. 

Cholera-red  reaction,  170. 

Cholesterin,  88,  90. 

Choline,  153. 

Coagulating  ferment,  15. 

Copper,  determination  in  copper  sul- 
phate, 176. 
sulphate,  estimation  of  sulphuric 

acid  in,  175. 
sulphate,  estimation  of  copper  in, 
176. 

Creatine,  21. 

Creatuiine,  22,  102. 

estimation  in  uruie,  192. 

Cresol,  106. 

estimation  in  urine,  195. 

Cresyl  sulphuric  acid,  107. 

Cystic  fluids,  64. 

Deniges,  reaction  of,  79. 
Dextrin,  74. 
Dextrose,  117. 
Deuteroalbumose,  43. 
Diabetic  sugar,  117. 
Diastase,  75. 

Digestion  experiments,  quantitative, 
240. 
influence  of  quantity  of  pepsin  on, 

37. 
interference  of  certain  substances 

with,  38. 
products  of,  39. 
Dysalbumose,  43. 

Egg,  yolk  of  the,  150. 
Egg-albumen,  155. 
Ethereal  sulphates,  detection  in  urine, 
125. 
sulphates,  estimation,  207. 

Fseces,  analysis  of,  211. 

estimation  of  carbohydrates  in,  213. 

estimation  of  fat  in,  212. 

estimation  of  nitrogen  in,  212. 

estimation  of  phosphorus  in,  214. 

estimation  of  sulphur  in,  216. 

estimation  of  water  in,  211. 
Fat,  141. 

estimation  in  blood,  232. 

estimation  in  bread,  229. 

estimation  in  feeces,  212. 

estimation  in  meat,  218. 

estimation  in  milk,  221. 

saponification  of,  142. 


Fat-splitting  ferment,  82. 
Fatty  acids,  143. 
Fehlmg's  solution,  198. 
Fermentation  tests,  13,  119. 
Fibrin,  conduct  of,  58. 
Fiirbringer,  detection  erf  mercury  la 
urine,  127. 

Galactose,  14. 
Gall-stones,  90. 
Gastric  digestion,  33. 

juice,  artificial,  40. 
Gelatin,  137. 
Globulm,  59. 
Glucose,  117. 

detection  in  urine,  118. 

detection  in  the  white  of  the  egg, 
157. 

estimation  in  urine,  196. 
Glutin,  137. 
Glycerin,  143,  148. 

phosphoric  acid,  153. 
Glycocholic  acid,  87. 
Glycogen,  131. 

estimation,  244. 
Gmelin's  reaction,  93,  124. 

reaction,     Rosenbach's     modifica- 
tion, 124. 
Grape-sugar,  117. 
Guanine,  136. 

Gumiing's  test  for  acetone,  121. 
Giinzburg's  reagent,  35. 

Hsematin,  53. 

hydrochloride,  54. 
Hi3ematoporphyrin,  56. 

detection  in  urine,  123. 
Hsemin,  54. 

test,  55. 
Heemochromogen,  53. 
Hsemoglobin,  51. 

Heller's  test  for  blood-pigments,  123. 
Heteroalbumose,  43. 
Hippui-ic  acid,  104. 
Hydrochloric  acid,  detection  in  gas- 
tric juice,  33. 

acid,   estimation   in  gastric  juice, 
237. 

acid,  estimation  in  urine,  205. 

acid,  standard  solution,  184. 
Hydrocinnamic  acid,  172. 
Hydroparacumaric  acid,  173. 
Hj^oxanthine,  28. 

Indican  test,  Jaffe's,  110. 


INDEX. 


261 


Indigo-blue,  109. 

Indigo-red,  110. 

Indigo  test  for  milk-sugar,  12. 

Indol,  169. 

Indoxylsulphuric  acid,  109. 

Iodide    of    potassium,    detection    in 

urine,  126. 
lodofonn  test  for  acetone,  120. 
Iron,  estimation  in  blood,  230. 

Jaffe's  reaction  for  mdican,  110. 
reaction  for  creatinine,  103. 

Kjeldahl  method  for  the  determina- 
tion of  nitrogen,  184. 

Lactic  acid  fermentation,  17. 
acid  detection,  35. 

Lactose,  10. 

Lassaigne  test,  6. 

Lecithin,  155. 

Legal' s  reaction  for  acetone,  120. 

Leucine,  80. 

Liebermann's    reaction    for    albu- 
min, 6. 

Liebennann-Burchard  reaction  for 
cholesterin,  92. 

Lipase,  82. 

Lipolytic  ferment,  82. 

Liver,  examination  of,  131. 

Lutein,  152. 

Maltose,  75. 

Meat,  analysis  of,  217. 

detection  of  proteids  in,  23. 
Mercury,  detection  in  urine,  127. 
Methsemoglobin,  52. 
Methyl  violet,  34. 
Milk,  action  of  rennm  on,  15. 

analysis  of,  221. 

coagulation  of,  15. 

general  properties  of ,  1 . 

separation  mto  its  constituents,  3. 
Milk-sugar,  10. 

conduct   on   heating   with   hydro- 
chloric acid,  14. 

estimation  in  milk,  225. 

reactions  of,  11. 
Millon's  reaction,  5. 
Molisch's  reaction  for  gluco.se,  118. 
Moore's  test  for  svigar,  1 1 . 
Mucic  acid,  14. 

Mucin,  detection  in  pathological  flu- 
ids, 64. 

detection  in  saliva,  70. 

of  the  bile,  88. 


Murexide  test,  101. 
Muscular  tissue,  20,  217. 
Myosin,  preparation,  24. 

Nitrogen,  detection  of,  6. 

estimation  in  blood,  231. 

estimation  in  bread,  229. 

estimation  m  fseces,  212. 

estimation  in  meat,  217. 

estimation  in  milk,  222. 

estimation  m  urine,  184. 
Nuclein,  135. 
Nucleoalbumin,  65. 
Nucleoproteid  of  the  pancreas,  83. 

Oleic  acid,  146. 

Orcin  test  for  pentose,  84. 

Ossein,  137. 

Ovalbumin,  155. 

Ovomucoid,  158. 

Oxalic  acid,  detection  in  urine,  104. 

acid,  estimation  in  urine,  194. 
Oxyha?moglobin,  preparation  of,  50. 
Oxyphenyl-acetic  acid,  172. 
Oxyphenyl-propionic  acid,  173. 

Pabnitic  acid,  147. 
Pancreas,  76. 

diastatic  action  of,  81. 
Paralbmnin,  detection  of,  68. 
Paracasein,  16 
Paracresol,  171. 
Paranuclein,  9,  154. 
Paranucleic  acid,  154. 
Paroxyphenyl-acetic  acid,  172. 
Paroxyphenyl-propionic  acid,  173. 
Pathological  transudates,  64. 
Pepsin,    commercial,    comparison   of 
the  different  kinds,  38. 

detection  of,  36,  113. 

digestion,  39. 

digestion,  disturbing  influences,  38, 
240. 
Peptone,  detection  in  urine,  115. 

Kiihne's,  46. 

preparation  of,  46. 
Pettenkofer's  reaction  for  the    bile 

acids,  86. 
Phenol,  106,  161. 

estimation  in  urine,  195. 
Phenyl-acetic  acid,  171. 
Phenyl-hydrazine   test   for    glucose, 

118. 
Phenyl-propionic  acid,  172. 


262 


INDEX. 


Phenyl-sulphuric  acid,  107. 
Phloroglucin  test  for  pentose,  84. 
Phosphates,  detection  in  urine,  125. 
Phosphoric  acid,  detection  in  urine, 
125. 

acid,  estimation  in  virine,  208. 
Phosphorus^  detection  of,  9. 

estimation  in  blood,  231. 

estimation  in  bread,  230. 

estimation  in  faeces,  214. 

estimation  in  meat,  219. 

estimation  in  milk,  225. 
Piria's  test,  79. 

Potassium     bromide,     detection     in 
urine,  126. 

iodide,  detection  in  urine,  126. 
Potassium  sulphocyanate,  detection, 

71. 
Protalbumose,  43. 
Proteids  of  the  blood,  59. 

of  meat,  23. 

of  milk,  3. 

of  milk,  estimation,  224. 

putrefaction  products  of,  159. 
Pseudomucin,  detection,  68. 
Ptyalin,  detection  of,  71. 

influence  of  acids  on  action  of,  72. 
Purine  bases,  24. 
Putrefaction    of    proteids,    products 

of,  159. 
PjTOcatechin,  109. 

Reagents,  list  of,  249. 
Rennin,  action  on  milk,  15 
Rubner's  test,  13. 

Saliva  and  salivary  digestion,  70. 

conduct  towards  reagents,  70. 
Salivary  digestion,  isolation  of  prod- 
ucts of,  73. 
Saponification  of  the  fats,  143. 
Sarcolactic  acid,  30. 
Serum  albumin,  reactions,  60,  156. 

albumin,  separation  from  globulin, 
59. 
Skatol,  170. 

Skatol-carbonic  acid,  173. 
Specific  gravities,  tables  of,  253. 
Stearic  acid,  147. 
Succinic  acid,  174. 
Sugar,  detection  in  blood,  57. 

detection  m  pathological  transu- 
dates, 68. 

detection  in  serous  fluids,  68. 


'  Sugar,  detection  in  the  egg,  157. 

detection  in  the  liver,  133. 

detection  in  the  urine  US. 

estimation  in  urine,  196. 
Siilphates,  detection  in  urine,  125. 
Sulphohsemoglobin,  52. 
Sulphur,  detection  of,  6. 

estimation  m  blood,  232. 

estimation  in  faeces,  216. 

estimation  in  meat,  219. 

estimation  in  milk,  228. 

estimation  in  urine,  207. 

unoxidized  in  urme,  1 13. 
Sulphuric  acid,  estimation  in  urine, 
205. 

determination  in  copper  sulphate, 
175. 

Taurine,  88. 
Taurocholic  acid,  87. 
Transudates,  64. 
Tryptic  digestion,  76. 
Trommer's  test,  11. 
Tyrosine,  78. 

UfTeknann's  reagent,  35. 
Urea,  97. 

detection    in    pathological    tran- 
sudates, 66. 

determination  according  to  Liebig, 
180. 

determination  in  urine,  193. 

preparation  of,  96. 
Uric  acid,  99. 

acid  estimation  in  urine,  188. 

acid,  reactions  of,  101. 
Urine,  conduct  towards  reagents,  95. 

determination  of  total  nitrogen  in, 
187. 

examination  of,  95. 
Urinary  calculi,  128. 
Urobilin,  111. 

Vitellir,  154. 

Water,  estimation  in  blood,  231. 

estimation  in  bread,  229. 

estimation  In  faeces,  211. 

estimation  in  meat,  217. 

estimation  in  milk,  221. 
Water  of  crystallization,  determina- 
tion in  copper  sulphate,  177. 
Weidel's  reaction,  30. 


INDEX. 


263 


Weyl's  reaction,  22,  103. 
Wliite  of  the  egg,  155. 

Xanthine,  29. 

bases  in  meat,  24. 


Xanthine,  bases  in  the  liver,  134. 
Xanthoproteic  reaction,  4. 
Xanthoprotein,  61. 

Yolk  of  the  egg,  150. 


INDEX  TO  THE  PLATE  OF  ABSORPTION  SPECTRA. 

No.  1.  Oxyhsenioglobin. 

No.  2.  Hsemoglobin. 

No.  3.  Methsemoglobin  (in  neutral,  or  faintly  alkaline,  solution). 

No.  4.  Heematin  in  acid  alcohol  solution. 

No.  5.  Reduced  hasmatin  in  alkaline  solution. 

No.  6.  Hsematoporphyrin  in  acid  solution. 

No.  7.  Urobilin. 


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Architectural  Iron  and  Steel 8vo,  3  jo 

Compound  Riveted  Girders  as  Applied  in  Buildings 8vo,  2  co 

Planning  and  Construction  of  High  OfBce  Buildings 8vo,  3  50 

Skeleton  Construction  in  Buildings 8vo,  3  00 

Briggs's  Modern  American  School  Buildings 8vo,  4  00 

Carpenter's  Heating  and  Ventilating  of  Buildings 8vo,  4  00 

Freitag's  Architectural  Engineering.     2d  Edition,  Rewritten 8vo,  3  50 

Fireproofing  of  Steel  Buildings 8vo,  2  50 

French  and  Ives's  Stereotomy 8vo,  2  50 

Gerhard's  Guide  to  Sanitary  House-inspection i6mo,  i  00 

Theatre  Fires  and  Panics i2mo,  i  50 

Holly's  Carpenters'  and  Joiners'  Handbook i8mo,  o   75 

Johnson's  Statics  by  Algebraic  and  Graphic  Methods 8vo,  2  00 

1 


Kidder's  Architect's  and  Builder's  Pocket-book.     Rewritten  Edition. 

i6mo, morocco,  5  00 

Merrill's  Stones  for  Building  and  Decoration 8vo,  5  00 

Monckton's  Stair-building 4to,  4  00 

Patton's  Practical  Treatise  on  Foundations , 8vo,  5  00 

Peabody's  Naval  Architecture 8vo,  7  50 

Siebert  and  Biggin's  Modern  Stone-cutting  and  Masonry Svo,  i  50 

Snow's  Principal  Species  of  Wood Svo,  3  50 

Sondericker's  Graphic  Statics  with  Applications  to  Trusses,  Beams,  and  Arches. 

Svo,  2  00 

Wait's  Engineering  and  Architectural  Jurisprudence Svo,  6  00 

Sheep,  6  50 
Law  of  Operations  Preliminary  to  Construction  in  Engineering  and  Archi- 
tecture   Svo,  5  00 

Sheep,  5  50 

Law  of  Contracts Svo,  3  00 

Wood's  Rustless  Coatings:   Corrosion  and  Electrolysis  of  Iron  and  Steel . .  .8vo,  4  00 

Woodbury's  Fire  Protection  of  Mills Svo,  2  50 

Worcester  and  Atkinson's  SmaU  Hospitals,  Establishment  and  Maintenance, 
Suggestions  for  Hospital  Architecture,  with  Plans  for  a  SmaU  Hospital. 

i2mo,  I  25 

The  World's  Columbian  Exposition  of  1S93 Large  4to,  i  00 

ARMY  AND  NAVY. 

Bernadou's  Smokeless  Powder,  Nitro-cellulose, and  the  Theory  of  the  Cellulose 

Molecule i2mo,  2  50 

*  Bruff's  Text-book  Ordnance  and  Gunnery Svo,  6  00 

Chase's  Screw  Propellers  and  Marine  Propulsion Svo,  3  00 

Craig's  Azimuth 4tOj,  3  50 

Crehore  and  Squire's  Polarizing  Photo-chronograph Svo,  3  00 

Cronkhite's  Gunnery  for  Non-commissioned  Officers 24mo,  morocco,  2  00 

*  Davis's  Elements  of  Law Svo,  2  50 

*  Treatise  on  the  Military  Law  of  United  States Svo,  7  00 

Sheep,  7  50 

De  Brack's  Cavalry  Outpost  Duties.     (Carr.) 24mo  morocco,  2  00 

Dietz's  Soldier's  First  Aid  Handbook i6mo,  morocco,  i  25 

*  Dredge's  Modern  French  Artillery 4to,  half  morocco,    15  00 

Durand's  Resistance  and  Propulsion  of  Ships Svo,  5  00 

*  Dyer's  Handbook  of  Light  Artillery i2mo,  3  00 

Eissler's  Modem  High  Explosives Svo,  4  00 

*  Fiebeger's  Text-book  on  Field  Fortification Small  Svo,  2  00 

Hamilton's  The  Gunner's  Catechism iSmo,  i  00 

*  Hoff's  Elementary  Naval  Tactics Svo,  i  50 

Ingalls's  Handbook  of  Problems  in  Direct  Fire Svo,  4  00 

*  BaUistic  Tables Svo,  i  50 

*  Lyons's  Treatise  on  Electromagnetic  Phenomena.   Vols.  I.  and  II. .  Svo„  each,  6  00 

*  Mahan's  Permanent  Fortifications.     (Mercur.) Svo,  half  morocco,  7  50 

Manual  for  Courts-martial i6nio.  morocco,  i  50 

*  Mercur's  Attack  of  Fortified  Places i2mo,  2  00 

*  Elements  of  the  Art  of  War Svo,  4  00 

Metcalf 's  Cost  of  Manufactures — And  the  Administration  of  Workshops,  Public 

and  Private Svo,  5  00 

*  Ordnance  and  Gunnery.     2  vols i2mo,  5  00 

Murray's  Infantry  Drill  Regulations iSmo.  paper,  10 

Peabody's  Naval  Architecture Svo,  7  50 

*  Phelps's  Practical  Marine  Surveying Svo,  2  50 

Powell's  Army  Oflicer's  Examiner i2mo,  4  00 

Sharpe's  Art  of  Subsisting  Annies  in  War iSmo,  morocco,  i  50 

2 


I 

SO 

3 

oo 

I 

oo 

2 

00 

3 

00 

3 

oo 

I 

50 

I 

so 

2 

so 

3 

SO 

4 

00 

2 

50 

•  Walke's  Lectures  on  Explosives 8vo  4  00 

•  Wheeler's  Siege  Operations  and  Military  Mining 8vo,  2  00 

Winthrop's  Abridgment  of  Military  Law l2mo,  2  50 

Woodhull's  Notes  on  Military  Hygiene i6mo,  i   50 

Young's  Simple  Elements  of  Navigation i6mo  morocco,  i  00 

Second  Edition,  Enlarged  and  Revised i6mo,  morocco,  2  00 

ASSAYING. 

Fletcher's  Practical  Instructions  in  Quantitative  Assaying  with  the  Blowpipe. 

i2mo,  morocco, 

Furman's  Manual  of  Practical  Assaying 8vo, 

Miller's  Manual  of  Assaying i2mo, 

O'DriscoU's  Notes  on  the  Treatment  of  Gold  Ores 8vo, 

Ricketts  and  Miller's  Notes  on  Assaying 8vo, 

Ulke's  Modern  Electrolytic  Copper  Refining 8vo, 

Wilson's  Cyanide  Processes i2mo, 

Chlorination  Process i2mo, 

ASTRONOMY. 

Comstock's  Field  Astronomy  for  Engineers 8vo, 

Craig's  Azimuth 4to, 

Doolittle's  Treatise  on  Practical  Astronomy 8vo, 

Gore's  Elements  of  Geodesy 8vo, 

Hayford's  Text-book  of  Geodetic  Astronomy 8vo,    3  00 

Merriman's  Elements  of  Precise  Surveying  and  Geodesy 8vo,    2  50 

•  Michie  and  Harlow's  Practical  Astronomy 8vo,    3  00 

•  White's  Elements  of  Theoretical  and  Descriptive  Astronomy i2mo,    2  00 

BOTANY. 

Davenport's  Statistical  Methods,  with  Special  Reference  to  Biological  Variation. 

i6mo,  morocco,  i  25 

Tbom^  and  Bennett's  Structural  and  Physiological  Botany i6mo,  2  25 

Westermaier's  Compendium  of  General  Botany.     (Schneider.) 8vo,  2  00 

CHEMISTRY. 

Mriance's  Laboratory  Calculations  and  Specific  Gravity  Tables i2mo,  1   25 

Allen's  Tables  for  Iron  Analysis 8vo,  3  00 

Arnold's  Compendium  of  Chemistry.     (MandeL) Small  8vo,  3  50 

Austen's  Notes  for  Chemical  Students xzmo,  i   50 

•  Austen  and  Langworthy.      The    Occurrence   of  Aluminium   in   Vegetable 

Products,  Animal  Products,  and  Natural  Waters 8vo,  2  00 

Bemadou's  Smokeless  Powder. — Witro-cellulose,  and  Theory  of  the  Cellulose 

Molecule i2mo,  2  50 

Bolton's  Quantitative  Analysis 8vo,  i  50 

•  Browning's  Introduction  to  the  Rarer  Elements 8vo,  I  50 

Brush  and  Penfield's  Manual  of  Determinative  Mineralogy 8vo,  4  00 

Classen's  Quantitative  Chemical  Analysis  by  Electrolysis.  (Boltwood.)  . . .  .8vo,  3  00 

Cohn's  Indicators  and  Test-papers i2mo,  2  00 

Tests  and  Reagents 8vo,  3  00 

Copeland's  Manual  of  Bacteriology,     (/n  preparation.) 

Craft's  Short  Course  in  Qualitative  Chemical  Analysis.  (Schaeflfer.).  . . .  i2mo,  1  SO 

Dolezalek's  Theory    of    the    Lead    Accumulator    (Storage    Battery).     (Von 

Ende) i2mo,  2  50 

Drechsel's  Chemical  Reactions.     (Merrill.) i2ino,  I  25 

Duhem's  Thermodynamics  and  Chemistry.     (Burgess.) 8vo,  4  00 

Eissler's  Modern  High  Explosives 8vo,  4  00 

SSront's  Enzymes  and  their  Applications.     (Prescott.) 8to,  3  00 

Brdmann'g  Introduction  to  Chemical  Preparations.     (Dtuilap.) i2mo,  i  2g 

3 


Fletcher's  Practical  Instructions  in  Quantitative  Assaying  with  the  Blowpipe 

i2mo,  morocco,  i  50 

Fowler's  Sewage  Works  Analyses i2mo,  2  00 

Fresenius's  Manual  of  Qualitative  Chemical  Analysis.     (Wells.) 8vo,  5  00 

Manual  of  Qualitative  Chemical  Analysis.     Parti.    Descriptive.     (Wells.) 

8vo,  3  00 
System   of  Instruction   in    Quantitative   Chemical  Analysis.      (Cohn.) 

2  vols 8vo,  12  50 

Fuertes's  Water  and  Public  Health i2mo,  i  so 

Furman's  Manual  of  Practical  Assaying 8vo,  3  00 

*Getman's  Exercises  in  Physical  Chemistry i2mo,  2  00 

Gill's  Gas  and  Fuel  Analysis  for  Engineers i2mo,  i  25 

Grotenfelt's  Principles  of  Modem  Dairy  Practice.     (Woll.) i2mo.  2  00 

Hammarsten's  Text-book  of  Physiological  Chemistry.     (MandeO 8vo,  4  00 

Helm's  Principles  of  Mathematical  Chemistry.     (Morgan.) i2mo,  i  50 

Hering's  Ready  Reference  Tables  (Conversion  Factors) i6mo,  morocco,  2  50 

Hinds's  Inorganic  Chemistry 8vo,  3  00 

•  Laboratory  Manual  for  Students i2mo,  75 

Holleman's  Text-book  of  Inorganic  Chemistry.     (Cooper.) 8vo,  2  50 

Text-book  of  Organic  Chemistry.     (Walker  and  Mott.) 8vo,  2  50 

•  Laboratory  Manual  o!  Organic  Chemistry.     (Walker.) i2mo,  i  00 

Hopkins's  Oil-chemists'  Handbook 8vo,  3  00 

Jackson's  Directions  for  Laboratory  Work  in  Physiological  Chemistry . .  8vo,  i  25 

Keep's  Cast  Iron 8v0r  2  50 

Ladd's  Manual  of  Quantitative  Chemical  Analysis , i2mo,  i  00 

Landauer's  Spectrum  Analysis.     (Tingle.) 8vo,  3  00 

Lassar-Cohn's  Practical  Urinary  Analysis.     (Lorenz.) i2mo,  1  00 

Leach's  The  Inspection  and  Analysis  of  Food  with  Special  Reference  to  State 

Control.     (In  preparation.) 

LSb's  Electrolysis  and  Electrosynthesis  of  Organic  Compounds.  (Lorenz.)  i2mo,  1  00 

Mandel's  Handbook  for  Bio-chemical  Laboratory i2mo,  i  50 

•  Martin's  Laboratory  Guide  to  Qualitative  Analysis  with  the  Blowpipe . .  i2mo,  60 
Mason's  Water-supply.     (Considered  Principally  from  a  Sanitary  Standpoint.) 

3d  Edition,  Rewritten 8vo,  4  00 

Examination  of  Water.     (Chemical  and  BacteriologicaL) i2mo,  i  25 

Meyer's  Determination  of  Radicles  in  Carbon  Compounds.     (Tingle.).  .i2mo,  i  00 

Miller's  Manual  of  Assaying i2mo,  i  00 

Milter's  Elementary  Text-book  of  Chemistry ." i2mo,  i  50 

Morgan's  Outline  of  Theory  of  Solution  and  its  Results i2mo,  i  00 

Elements  of  Physical  Chemistry i2mo,  2  00 

Morse's  Calculations  used  in  Cane-sugar  Factories i6mo,  morocco,  i  50 

Mulliken's  General  Method  for  the  Identification  of  Pure  Organic  Compounds. 

VoL  I Large  8vo,  5  00 

Nichols's  Water-supply.     (Considered  mainly  from  a  Chemical  and  Sanitary 

Standpoint,  1883.) 8vo,  2  50 

O'Biine's  Laboratory  Guide  in  Chemical  Analysis 8vo,  2  00 

O'Driscoll's  Notes  on  the  Treatment  of  Gold  Ores 8vo,  2  00 

Ost  and  Kolbeck's  Text-book  of  Chemical  Technology.     (Lorenz — Bozart.) 

(In  preparation.) 
Ostwald's  School  of  Chemistry.     Part  One.     (Ramsey.)     (In  press.) 

•  Penfield's  Kotes  on  Determinative  Mineralogy  and  Record  of  Mineral  Tests. 

8vo,  paper,  50 

Pictet's  The  Alkaloids  and  their  Chemical  Constitution.     (Biddle.) 8vo,  5  00 

Pinner's  Introduction  to  Organic  Chemistry.     (Austen.) i2mo,  i  50 

Pools's  Calorific  Power  of  Fuels 8vo,  3  00 

Prescott  and  Winslow's  Elements  of  Water  Bacteriology,  with  Special  Refer- 
ence to  Sanitary  Water  Analysis i2mo,  i   2S 

•  Reisig's  Guide  to  Piece-dyeing 8vo,  25  00 

4 


Richards  and  Woodman's  Air  .Water,  and  Food  from^  Sanitary  Standpoint.  8vo,  2  00 

Richards's  Cost  of  Living  as  Modified  by  Sanitary  Science i2mo,  i  00 

Cost  of  Food  a  Study  in  Dietaries i2mo,  i  00 

•  Richards  and  Williams's  The  Dietary  Computer 8vo,  i  50 

Ricketts  and  Russell's  Skeleton  Notes  upon  Inorganic  Chemistry.     (Part  I. — 

Non-metallic   Elements.) 8vo,  morocco,  75 

Ricketts  and  Miller's  Notes  on  Assaying Svo,  3  00 

Rideal's  Sewage  and  the  Bacterial  Purification  of  Sewage Svo,  3  50 

Disinfection  and  the  Preservation  of  Food Svo,  4  00 

Ruddiman's  Incompatibilities  in  Prescriptions Svo,  2  00 

Sabin's  Industrial  and  Artistic  Technology  of  Vaints  and  Varnish.     {In  press.) 

Salkowski's  Physiological  and  Pathological  Chemistry.     (Orndorff.*). .  .  .Svo,  2  50 

Schimpf's  Text-book  of  Volumetric  Analysis i2mo,  2  50 

Essentials  of  Volumetric  Analysis t2mo,  I  25 

Spencer's  Handbook  for  Chemists  of  Beet-sugar  Houses i6mo,  morocco,  3  00 

Handbook  for  Sugar^anufacturers  and  their  Chemists.  .i6mo,  morocco,  3  00 

Stockbridge's  Rocks  and  Soils Svo,  3  S« 

•  Tillman's  Elementary  Lessons  in  Heat Svo,  i  50 

•  Descriptive  General  Chemistry Svo,  3  00 

Treadwell's  Qualitative  Analysis.     (Hall.) Svo,  3  00 

Quantitative  Analysis.     (Hall.) Svo,  4  00 

Tumeaure  and  Russell's  Public  "Water-supplies Svo,  5  00 

Van  Deventer's  Physical  Chemistry  for  Beginners.     (Boltwood.) i2mo,  i  50 

•  Walke's  Lectures  on  Explosives Svo,  4  00 

Wassermann's  Immune  Sera:  Haemolysins,  Cytotoxins,  and  Precipitins.     (Bol- 

duan.) i2mo,  i  00 

Wells's  Laboratory  Guide  in  Qualitative  Chemical  Analysis Svo,  i  50 

Short  Course  in  Inorganic  Qualitative  Chemical  Analysis  for  Engineering 

Students i2mo,  i  50 

Whipple's  Microscopy  of  Drinking-water Svo,  3  50 

Wiechmann's  Sugar  Analysis Small  Svo.  2  50 

Wilson's  Cyanide  Processes i2mo,  i  50 

Chlorination  Process i2mo,  i  50 

WuUing's  Elementary  Course  in  Inorganic  Pharmaceutical  and  Medical  Chem- 
istry  i2mo,  2  00 

CIVIL  ENGINEERING. 

BRIDGES  AND    ROOFS.       HYDRAULICS.      MATERIALS    OF    ENGINEERING 
RAILWAY  ENGINEERING, 

Baker's  Engineers'  Surveying  Instruments i2mo,  3  00 

Bixby's  Graphical  Computing  Table Paper  19^X24}  inches.  35 

*•  Burr's  Ancient  and  Modem  Engineering  and  the  Isthmian  Canal.     (Postage, 

27  cents  additionaL) Svo,  net,  3  50 

Comstock's  Field  Astronomy  for  Engineers Svo,  2  50 

Davis's  Elevation  and  Stadia  Tables Svo,  I  00 

Elliott's  Engineering  for  Land  Drainage i2mo,  I  50 

Practical  Farm  Drainage i2mo,  I  00 

Folwell's  Sewerage.     (Designing  and  Maintenance.) Svo,  3  00 

Freitag's  Architectural  Engineering.     2d  Edition,  Rewritten Svo,  3  90 

French  and  Ives's  Stereotomy Svo,  3  50 

Goodhue's  Municipal  Improvements i2mo.  I  75 

Goodrich's  Economic  Disposal  of  Towns'  Refuse Svo,  3  50 

Gore's  Elements  of  Geodesy Svo,  2  50 

Hayford's  Text-book  of  Geodetic  Astronomy Svo,  3  o<r 

Hering's  Ready  Reference  Tables  (Conversion  Factors) i6mo,  morocco,  2  50 

Howe's  Retaining  Walls  for  Earth i2mo,  i   25 

Johnson's  Theory  and  Practice  of  Surveying Small  Svo,  4  00 

Statics  by  Algebraic  and  Graphic  Methods Svo,  z  00 

5 


s 

oo 

I 

so 

2 

SO 

2 

00 

2 

oo 

3 

50 

2 

oo 

7 

so 

5 

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3 

5t 

I 

5c 

2 

50 

2 

00 

5 

00 

6 

00 

6 

50 

5 

00 

5 

50 

3 

00 

Eiersted's  Sewage  Disposal i2mo,    i  25 

Laplace's  Philosophical  Essay  on  Probabilities.     (Truscott  and  Emory.)  i2mo,    2  00 
Mahan's  Treatise  on  Civil  Engineering.     (1873.)     (Wood.) 8vo, 

•  Descriptive  Geometry 8vo, 

Merriman's  Elements  of  Precise  Surveying  and  Geodesy 8vo, 

Elements  of  Sanitary  Engineering 8vo, 

Merriman  and  Brooks's  Handbook  for  Surveyors i6mo,  morocco, 

Nugent's  Plane  Surveying ., 8vo, 

Ogden's  Sewer  Design i2mo, 

Patton's  Treatise  on  Civil  Engineering 8vo  half  leather. 

Reed's  Topographical  Drawing  and  Sketching 4to, 

Rideal's  Sewage  and  the  Bacterial  Purification  of  Sewage 8vo, 

Siebert  and  Biggin's  Modem  Stone-cutting  and  Masonry 8vo, 

Smith's  Manual  of  Topographical  Drawing.     (McMillan.) 8vo, 

Sondericker's  Graphic   Statics,  wun  Applications   to  Trusses,  Beams,   and 
Arches 8vo, 

*  Trautwine's  Civil  Engineer!s  Pocket-book i6mo,  morocco. 

Wait's  Engineering  and  Architectural  Jurisprudence 8vo, 

Sheep, 
Law  of  Operations  Preliminary  to  Construction  in  Engineering  and  Archi- 
tecture.  8vo, 

Sheep, 

Law  of  Contracts 8vo, 

Warren's  Stereotomy — Problems  in  Stone-cutting 8vo,    2  50 

Webb's  Problems  in  the  Use  and  Adjustment  of  Engineering  Instruments. 

i6mo,  morocco,    i  25 

•  Wheeler's  Elementary  Course  of  Civil  Engineering 8vo,    4  00 

Wilson's  Topographic  Surveying , .,,,,,,  ,8vo,    3  50 

BRIDGES  AND  ROOFS. 

Boiler's  Practical  Treatise  on  the  Construction  of  Iron  Highway  Bridges.  .8vo,    2  00 

*  Thames  River  Bridge 4to,  paper,    5  00 

Burr's  Course  on  the  Stresses  in  Bridges  and  Roof  Trusses,  Arched  Ribs,  and 

Suspension  Bridges 8vo,  3  50 

Du  Bois's  Mechanics  of  Engineering.     VoL  II Small  4to,    10  00 

Foster's  Treatise  on  Wooden  Trestle  Bridges 4to,  5  00 

Fowler's  Coffer-dam  Process  for  Piers 8vo,  2  50 

Greene's  Roof  Trusses 8vo,  i  25 

Bridge  Trusses 8vo,  2  50 

Arches  in  Wood,  Iron,  and  Stone 8vo,  2  50 

Howe's  Treatise  on  Arches 8vo,  4  00 

Design  of  Simple  Roof-trusses  in  Wood  and  Steel 8vo,  2  00 

J«hnson,  Bryan,  and  Tumeaure's  Theory  and  Practice  in  the  Designing  of 

Modem  Framed  Structures Small  4to,    10  00 

Merriman  and  Jacoby's  Text-book  on  Roofs  and  Bridges: 

Part  I. — Stresses  in  Simple  Trusses 8vo,  2  50 

Part  n. — Graphic  Statics 8vo,  2  50 

Part  in. — Bridge  Design.    4th  Edition,  Rewritten 8vo,  2  50 

Part  IV. — Higher  Structures 8vo,  2  50 

Morison's  Memphis  Bridge 4to,  10  00 

Waddell's  De  Pontibus,  a  Pocket-book  for  Bridge  Engineers. . .  i6mo,  morocco,  3  00 

Specifications  for  Steel  Bridges i2mo,  i  25 

Wood's  Treatise  on  the  Theory  of  the  Construction  of  Bridges  and  Roofs. 8vo,  2  00 
Wright's  Designing  of  Draw-spans: 

Part  I.  — Plate-girder  Draws Svo,  2  50 

Part  II. — Riveted-truss  and  Pin-connected  Long-span  Draws 8vo,  2  50 

Two  parts  in  one  volume Svo,  3  50 

6 


HYDRAULICS. 

Bazin's  Experiments  upon  the  Contraction  of  the  Liquid  Vein  Issuing  from  an 

Orifice.     (Trautwine.) 8vo,  2  00 

Bovey's  Treatise  on  Hydraulics 8vo,  5  00 

Church's  Mechanics  of  Engineering 8vo,  6  00 

Diagrams  of  Mean  Velocity  of  Water  in  Open  Channels paper,  i  50 

Coffin's  Graphical  Solution  of  Hydraulic  Problems i6mo,  morocco,  2  50 

Flather's  Dynamometers,  and  the  Measurement  of  Power i2mo,  3  00 

Folwell's  Water-supply  Engineering Svo,  4  00 

Frizell's  Water-power Svo,  5  00 

Fuertes's  Water  and  Public  Health i2mo,  1  50 

Water-filtration  Works i2mo,  2  50 

GanguUlet  and  Kutter's  General  Formula  for  the  Uniform  Flow  of  Water  Ln 

Rivers  and  Other  Channels.     (Hering  and  Trautwine.) Svo,  4  00 

Hazen's  FUtration  of  Public  Water-supply Svo,  3  00 

Hazlehurst's  Towers  and  Tanks  for  Water- works Svo,  2  50 

Herschel's  115  Experiments  on  the  Carrying  Capacity  of  Large,  Riveted,  Metal 

Conduits Svo,  2  00 

Mason's   Water-supply.     (Considered    Principally   from   a   Sanitary   Stand- 
point.)    3d  Edition,  Rewritten Svo,  4  00 

Merriman's  Treatise  on  Hydraulics,     gth  Edition,  Rewritten Svo,  5  00 

♦  Michie's  Elements  of  Analytical  Mechanics Svo,  4  00 

Schuyler's  Reservoirs  for  Irrigation,  Water-power,  and  Domestic   Water- 
supply  Large  Svo,  5  00 

•*  Thomas  and  Watt's  Improvement  of  Riyers.     (Post.,  44  c.  additional),  4to,  6  00 

Tumeaure  and  Russell's  Public  Water-supplies Svo,  5  00 

Wegmann's  Desien  and  Construction  of  Dams 4to,  5  00 

Water-supply  of  the  City  of  New  York  from  1658  to'iSgS 4to,  10  00 

Weisbach's  Hydraulics  and  Hydraulic  Motors.     (Du  Bois.) Svo,  5  00 

Wilson's  Manual  of  Irrigation  Engineering Small  Svo,  4  00 

Wolff's  Windmill  as  a  Prime  Mover Svo,  3  00 

Wood's  Turbines Svo,  3  50 

Elements  of  Analjrtical  Mechanics Svo,  3  00 

MATERIALS  OF  ENGINEERING. 

Baker's  Treatise  on  Masonry  Construction Svo,  5  00 

Roads  and  Pavements Svo,  5  00 

Black's  United  States  Public  Works Oblong  4to,  5  00 

Bovey's  Strength  of  Materials  and  Theory  of  Structures Svo,  7  50 

Burr's  Elasticity  and  Resistance  of  the  Materials  of  Engineering.     6th  Edi- 
tion, Rewritten Svo,  7  50 

Byrne's  Highway  Construction Svo,  5  00 

Inspection  of  the  Materials  and  Workmanship  Employed  in  Construction. 

i6mo,  3  00 

Church's  Mechanics  of  Engineering Svo,  6  00 

Du  Bois's  Mechanics  of  Engineering.     VoL  I Small  4to,  7  50 

Johnson's  Materials  of  Construction Large  Svo,  6  00 

Keep's  Cast  Iron Svo,  2  50 

Lanza's  Applied  Mechanics Svo,  7  50 

Martens's  Handbook  on  Testing  Materials.     (Henning.)     2  vols Svo,  750 

Merrill's  Stones  for  Building  and  Decoration Svo,  5  00 

Merriman's  Text-book  on  the  Mechanics  of  Materials Svo,  4  00 

Strength  of  Materials i2mo,  i  00 

Metcalf's  Steel.     A  Manual  for  Steel-users i2mo,  2  00 

Patton's  Practical  Treatise  on  Foundations Svo,  5  00 

7 


Rockwell's  Roads  and  Pavements  in  France lamo,  i  25 

Smith's  Materials  of  Machines i2mo,  i  00 

Snow's  Principal  Species  of  Wood 8vo,  3  50 

Spalding's  Hydraulic  Cement i2mo,  2  00 

Text-book  on  Roads  and  Pavements i2mo,  2  00 

Thurston's  Materials  of  Engineering.     3  Parts 8vo,  8  00 

Part  I. — Non-metallic  Materials  of  Engineering  and  Metallurgy Svo,  2  00 

Part  n.— Iron  and  Steel Svo,  3  50 

Part  in. — A  Treatise  on  Brasses,  Bronzes,  and  Other  Alloys  and  their 

Constituents Svo,  2  50 

Thurston's  Text-book  of  the  Materials  of  Construction Svo,  5  00 

Tillson's  Street  Pavements  and  Paving  Materials _, Svo,  4  00 

Waddell's  De  Pontibus.     (A  Pocket-book  for  Bridge  Engineers.). .  i6mo,  mor.,  3  00 

Specifications  for  Steel  Bridges i2mo,  i  25 

Wood's  Treatise  on  the  Resistance  of  Materials,  and  an  Appendix  on  the  Pres- 
ervation of  Timber Svo,  2  00 

Elements  of  Analytical  Mechanics Svo,  3  00 

Wood's  Rustless  Coatings:  Corrosion  and  Electrolysis  of  Iron  and  Steel.  .  .Svo,  4  00 

RAILWAY  ENGINEERING. 

Andrews's  Handbook  for  Street  Railway  Engineers.     3X5  inches,  morocco,  i  25 

Berg's  Buildings  and  Structures  of  American  Railroads 4to,  5  00 

Brooks's  Handbook  of  Street  Railroad  Location i6mo.  morocco,  i  50 

Butts's  Civil  Engineer's  Field-book i6mo,  morocco,  2  50 

Crandall's  Transition  Curve i6mo,  morocco,  i  50 

Railway  and  Other  Earthwork  Tables Svo,  i  50 

Dawson's  "Engineering"  and  Electric  Traction  Pocket-book.    i6mo,  morocco,  5  00 

Dredge's  History  of  the  Pennsylvania  Railroad:    (1S79) Paper,  5  00 

•  Drinker's  Tunneling,  Explosive  Compounds,  and  Rock  Drills,  4to,  half  mor.,  25  00 

Fisher's  Table  of  Cubic  Yards Cardboard,  25 

Godwin's  Railroad  Engineers'  Field-book  and  Explorers'  Guide i6mo,  mor.,  2  50 

Howard's  Transition  Curve  Field-book i6mo,  morocco,  i  50 

Hudson's  Tables  for  Calculating  the  Cubic  Contents  of  Excavations  and  Em- 
bankments    Svo,  I  00 

Molitor  and"  Beard's  Manual  for  Resident  Engineers i6mo,  1  00 

Kagle's  Field  Manual  for  Railroad  Engineers i6mo,  morocco.  3  00 

Philbrick's  Field  Manual  for  Engineers i6mo,  morocco,  3  00 

Searles's  Field  Engineering i6mo,  morocco,  3  00 

Railroad  Spiral i6mo,  morocco,  i  50 

Taylor's  Prismoidal  Formulae  and  Earthwork Svo,  1  50 

•  Trautwine's  Method  of  Calculating  the  Cubic  Contents  of  Excavations  and 

Embankments  by  the  Aid  of  Diagrams Svo,  2  00 

The  Field  Practice  of  [Laying    Out    Circular    Curves    for    Railroads. 

i2mo,  morocco,  2  50 

Cross-section  Sheet Paper,  25 

Webb's  Railroad  Construction.     2d  Edition,  Rewritten i6rno.  morocco,  5  00 

Wellington's  Economic  Theory  of  the  Location  of  Railways Small  Svo,  5  00 

DRAWING. 

Barr's  Kinematics  of  Machinery Svo,    2  50 

•  Bartlett's  Mechanical  Drawing Svo,    3  00 

•  "  '  "         Abridged  Ed Svo,    i  50 

Coolidge's  Manual  of  Drawing Svo,  paper,    i  00 

Coolidge  and  Freeman's  Elements  of  General  Drafting  for  Mechanical  Engi- 
neers.    {In  press.) 

Durley's  Kinematics  of  Machines Svo,    4  00 

8 


3 

oo 

3 

00 

5 

00 

4 

oo 

I 

50 

I 

50 

3 

50 

5 

00 

3 

00 

3 

oo 

3 

00 

2 

50 

I 

00 

I 

25 

I 

50 

I 

oo 

Z 

25 

75 

3 

50 

3 

00 

7 

50 

2 

50 

Hill's  Text-book  on  Shades  and  Shadows,  and  Perspective 8vo,    2  oo 

Jamison's  Elements  of  Mechanical  Drawing.     (In  press.) 

Jones's  Machine  Design: 

Part  I. — Kinematics  of  Machinery gyo     j  -p 

Part  n.— Form,  Strength,  and  Proportions  of  Parts 8vo', 

MacCord's  Elements  of  Descriptive  Geometrj  ,    , gvo 

Kinematics;   or.  Practical  Mechanism gvo 

Mechanical  Drawing ,    , .  j^ 

Velocity  Diagrams gy^ 

•  Mahan's  Descriptive  Geometry  and  Stone-cutting gvo. 

Industrial  Drawing.    (Thompson.) gyo 

Reed's  Topographical  Drawing  and  Sketching 4to, 

Reid's  Course  in  Mechanical  Drawing gyo 

Text-book  of  Mechanical  Drawing  and  Elementary  Machine  Design.  .8vo. 

Robinson's  Principles  of  Mechanism gyo 

Smith's  Manual  of  Topographical  Drawing.     (McMillan.) 8vo', 

Warren's  Elements  of  Plane  and  SoUd  Free-hand  Geometrical  Drawing.  .  i2mo! 

Drafting  Instruments  and  Operations i2mo. 

Manual  of  Elementary  Projection  Drawing i2mo. 

Manual  of  Elementary  Problems  in  the  Linear  Perspective  of  Form  and 

Shadow j2ino^ 

Plane  Problems  in  Elementary  Geometry i2mo. 

Primary  Geometry ,    jjmo' 

Elements  of  Descriptive  Geometry,  Shadows,  and  Perspective 8vo, 

General  Problems  of  Shades  and  Shadows 8to, 

Elements  of  Machine  Construction  and  Drawing 8vo 

Problems.  Theorems,  and  Examples  in  Descriptive  Geometry 8vo, 

Weisbach's  Kinematics  and  the  Power  of  Transmission.       (Hermann  and 

^ein.)  gyo^    5  ^^ 

Whelpley's  Practical  Instruction  in  the  Art  of  Letter  Engraving i2mo,    2  00 

Wilson's  Topographic  Surveying gyo^    3  5o 

Free-hand  Perspective '..*.".'.'.', 8vo'    a  50 

Free-hand  Lettering .' .' .  8vo.'    i  00 

Woolf'B  Elementary  Course  in  Descriptive  Geometry Large  8vo,    3  00 

ELECTRICITY  AND  PHYSICS. 

Anthony  and  Brackett's  Text-book  of  Physics.     (Magie.) SmaD  8vo,  3  00 

Anthony's  Lecture-notes  on  the  Theory  of  Electrical  Measurements . . .     i2mo*  i  00 

Benjamin's  History  of  Electricity "    o„..'  ,  „„ 

Voltaic  CelL y^y^y.'.'.'.'.'.]'.]'..'. 8vo'  00 

Ctassen's  Quantitative  Chemical  Analysis  by  Electrolysk.*  * (Boit^ood.). . 8vo'.  3  00 

Crehore  and  Sauier's  Polarizing  Photo-chronograph gyo'  ,  ^j, 

Diwson's"Eneineering"and  Electric  Traction  Pocket-book.' .iemo".  morocco'  5  00 
Dolezalek's    Theory  of    the    Lead   Accumulator    (Storage    Battery).     (Von' 

Ende.) 

TV,  .       ,   ^.            ,          '.       i2mo,  2  so 

Duhem  s  Thermodynamics  and  Chemistry.     (Burgess.) gyo  .   ^^ 

FUther's  Dvnamometers,  and  the  Measurement  of  Power i2mo'    3  00 

GUbert's  De  Magnete.     (Mottelay.) .*..'.*...."...   8vo'    2  so 

Hanchett's  Alternating  Currents  Explained * i2mo'    i  00 

Bering's  Ready  Reference  Tables  (Conversion  Factors)'.' .'.'.'.'.'i6mo',  morocco',    2  50 

Holman's  Precision  of  Measurements gy   ' 

Telescopic  Mirror-scale  Method.  Adjusttnents,  and  Tests.! . .  .Large  8vo '        75 

Landauer's  Spectrum  Analysis.    (Tingle. ) gyo' 

Le  ChateUer's  High-temperature  Measurements.  (Boudouard— Utirgess  )i 
Lab's  Electrolysis  and  Electrosynthesis  of  Organic  Compounds.  (Lorenz.)  lamo,    i 
•  Lyons'8  Treatise  on  Electromagnetic  Phenomena.     Vols.  I.  and  H.  8vo,  each,'    6 
•Michie.     ElemenU  of  Wave  Motion  RelaUng  to  Sound  and  Light.        »!8vo' 

9  


3 
i2mo,    3 


4 


7 

oo 

7 

50 

I 

SO 

6 

oo 

6 

50 

5 

00 

5 

so 

3 

00 

Niaudct's  Elementary  Treatise  on  Electric  Batteries.     (Fishoack. ) i2mo,  2  so 

•  Rosenberg's  Electrical  Engineering.    (Haldane  Gee — Kiflzbrunner.) 8vo,  i  50 

Ryan,  Norris,  and  Hoxie's  Electrical  Machinery.     Vol.  I 8vo,  2  50 

Thurston's  Stationary  Steam-engines 8vo,  2  50 

*  Tillman's  Elementary  Lessons  in  Heat 8vo,  i  50 

Tory  and  Pitcher's  Manual  of  Laboratory  Physics Small  8vo,  2  00 

Hike's  Modern  Electrolytic  Copper  Refining 8vo,  3  00 


LAW. 

•  Davis's  Elements  of  Law 7 8vo,    2  50 

•  Treatise  on  the  Military  Law  of  United  States 8vo, 

•  Sheep, 

Manual  for  Courts-martial i6mo,  morocco. 

Wait's  Engineering  and  Architectural  Jurisprudence 8vo, 

Sheep, 
Law  of  Operations  Preliminary  to  Construction  in  Engineering  and  Archi- 
tecture      8vo, 

Sheep, 

Law  of  Contracts 8vo, 

Winthrop's  Abridgment  of  Military  Law i2mo,    2  50 

MANUFACTURES. 

Bernadou's  Smokeless  Powder — Nitro-cellulose  and  Theory  of  the  Cellulose 

Molecule i2mo,    2  so 

Bolland'slron Founder i2mo,    2  so 

"  The  Iron  Founder,"  Supplement i2mo,    2  so 

Encyclopedia  of  Founding  and  Dictionary  of  Foundry  Terms  Used  in  the 

Practice  of  Moulding i2mo,    3  00 

Eissler's  Modem  High  Explosives 8vo,    4  00 

Effront's  Enzymes  and  their  Applications.     (Prescott.) 8vo,    3  00 

Fitzgerald's  Boston  Machinist i8mo,    i  00 

Ford's  Boiler  Making  for  Boiler  Makers i8mo,    1  00 

Hopkins's  Oil-chemists'  Handbook 8vo,   3  00 

Keep's  Cast  Iron 8vo,    2  so 

Leach's  The  Inspection  and  Analysis  of  Food  with  Special  Reference  to  State 

ControL     (.In  preparation.) 

Metcalf's  SteeL    A  Manual  for  Steel-users i2mo,    3  00 

Metcalfe's  Cost  of  Manufacttires — And  the  Administration    of  "Workshops, 

Public  and  Private 8vo,    5  00 

Meyer's  Modern  Locomotive  Construction 4to,  10  00 

Morse's  Calculations  used  in  Cane-sugar  Factories i6mo,  morocco,     1  50 

•  Reisig's  Guide  to  Piece-dyeing 8vo,  25  00 

Smith's  Press-working  of  Metals 8vo,    3  00 

Spalding's  Hydraulic  Cement i2mo,    2  00 

Spencer's  Handbook  for  Chemists  of  Beet-sugar  Houses i6mo,  morocco,    3  00 

HandbooK  tor  sugar  Manufacturers  ana  their  Chemists..  .i6mo,  morocco,  2  00 
Thurston's  Manual  of  Steam-boilers,  their  Designs,  Construction  and  Opera- 
tion  8vo,  5  00 

•  Walke's  Lectures  on  Explosives 8vo,  4  00 

West's  American  Foundry  Practice i2mo,  2  so 

Moulder's  Text-book i2mo,  3  50 

Wiechmann's  Sugar  Analysis Small  8vo,  2  50 

Wolff's  Windmill  as  a  Prime  Mover 8vo,  3  00 

Woodbury's J|ire  Protection  of  Mills 8vo,  2  50 

Wood's  Rustless  Coatings:   Corrosion  and  Electrolysis  of  Iron  and  Steel.  .  .8vo,  4  00 

10 


MATHEMATICS. 

Baker's  Elliptic  Functions 8vo,  i  go 

*  Bass's  Elements  of  Differential  Calculus ismo,  4  00 

Briggs's  Elements  of  Plane  Analytic  Geometry i2mo,  i  00 

Compton's  Manual  of  Logarithmic  Computations i2mo,  i  50 

Daris's  Introduction  to  the  Logic  of  Algebra 8vo,  i  50 

*  Dickson's  College  Algebra Large  i2mo,  I  50 

*  Answers  to  Dickson's  College  Algebra 8vo,  paper,  2S 

•  Introduction  to  the  Theory  of  Algebraic  Equations   Large  i2mo,  1  25 

Halsted's  Elements  of  Geometry 8vo,  i  75 

Elementary  Synthetic  Geometry 8vo,  i  50 

Rational  Geometry i2mo,  1   75 

•Johnson's  Three-place  Logarithmic  Tables:    Vest-pocket  size paper,  15 

100  copies  for  5  00 

•  Mounted  on  heavy  cardboard,  8  X  10  inches,  35 

10  copies  for  2  00 

Elementary  Treatise  on  the  Integral  Calculus Small  8vo,  i  50 

Curve  Tracing  in  Cartesian  Co-ordinates i2mo,  i  00 

Treatise  on  Ordinary  and  Partial  DiSerential  Equations Small  8vo,  3  50 

Theory  of  Errors  and  the  Method  of  Least  Squares i2mo,  i  50 

•  Theoretical  Mechanics i2mo,  3  00 

Laplace's  Philosophical  Essay  on  Probabilities.     (Truscott  and  Emory.)  i2mo,  2  00 

•  Ludlow  and  Bass.     Elements  of  Trigonometry  and  Logarithmic  and  Other 

Tables 8vo,  3  00 

Trigonometry  and  Tables  published  separately Each,  2  00 

*  Ludlow's  Logarithmic  and  Trigonometric  Tables 8vo,  i  00 

Maurer's  Technical  Mechanics 8vo,  4  00 

Merriman  and  Woodward's  Higher  Mathematics 8vo,  5  00 

Merriman's  Method  of  Least  Squares 8vo,  2  00 

Rice  and  Johnson's  Elementary  Treatise  on  the  Differential  Calculus. Sm.,  8vo,  3  00 

Differential  and  Integral  Calculus.     2  vols,  in  one Small  8vo,  2  50 

Sabin's  Industrial  and  Artistic  Technology  of  Paints  and  Varnish.     {In  press.) 

Wood's  Elements  of  Co-ordinate  Geometry 8vo,  2  00 

Trigonometry:  Analytical,  Plane,  and  Spherical i2mo,  i  00 

MECHANICAL   ENGINEERING. 
MATERIALS  OF  ENGINEERING,  STEAM-ENGINES  AND  BOILERS. 

Baldwin's  Steam  Heating  for  Buildings i2mo,  2  'So 

Barr's  Kinematics  of  Machinery ' 8vo,  2  50 

•  Bartlett's  Mechanical  Drawing 8vo,  3  00 

♦  "                 "               "        Abridged  Ed 8vo ,  i  50 

Benjamin's  Wrinkles  and  Recipes i2mo,  2  00 

Carpenter's  Experimental  Engineering 8vo,  6  00 

Heating  and  Ventilating  Buildings 8vo,  4  00 

Gary's  Smoke  Suppression  in  Plants  using  Bituminous  CoaL  _    (In  prep- 
aration.) 

Clerk's  Gas  and  Oil  Engine Small  8vo,  4  00 

Coolidge's  Manual  of  Drawing 8vo,    paper,  i   00 

Coolidge  and  Freeman's  Elements  of  General  Drafting  for  Mechanical  En- 
gineers.     (/((  jirrsK.) 

Cromwell's  Treatise  on  Toothed  Gearing i2mo.  i  50 

Treatise  on  Belts  and  PuLeys 1 2mo,  1  50 

Durley's  Kinematics  of  Machines 8vo,  4  00 

Flather's  Dynamometers  and  the  Measurement  of  Power i2mo,  3  00 

Rope  Driving i2mo,  2  00 

11 


OiU's  Gas  and  Fuel  Analysis  for  Engineers i2mo,  i  25 

Hall's  Car  Lubrication lamo,  i  00 

Bering's  Ready  Reference  Tables  (Conversion  Factors) i6mo,  morocco,  2  50 

Button's  Ttie  Gas  Engine 8vo,  s  oo 

Jones's  Machine  Design: 

Part  I. — Kinematics  of  Machinery 8vo,  1  50 

Part  n. — Form,  Strength,  and  Proportions  of  Parts 8vo,  3  00 

Kent's  Mechanical  Engineer's  Pocket-book i6mo,    morocco,  5  00 

Kerr's  Power  and  Power  Transmission 8vo,  2  00 

MacCord's  Kinematics ;  or.  Practical  Mechanism. . .' 8vo,  5  00 

Mechanical  Drawing 4to,  4  00 

Velocity  Diagrams r 8vo,  i  so 

Mahan's  Industrial  Drawing.    (Thompson.) 8vo,  3  50 

Poole's  Calorific  Power  of  Fuels 8vo,  3  00 

Reid's  Course  in  Mechanical  Drawing 8vo,  2  00 

Text-book  of  Mechanical  Drawing  and  Elementary  Machine  Design . .  8vo,  3  00 

Richards's  Compressed  Air I2m0t  i  50 

Robinson's  Principles  of  Mechanism 8vo,  3  00 

Smith's  Press-working  of  Metals .8vo,  3  00 

Thurston's  Treatise  on   Friction  and    Lost  Work   in   Machinery   and   Mill 

Work 8vo,  3  00 

Animal  as  a  Machine  and  Prime  Motor,  and  the  Laws  of  Energetics .  i2mo,  i  00 

Warren's  Elements  of  Machine  Construction  and  Drawing 8(ro,  V  so 

Weisbach's  Kanematics  and  the  Power  of  Transmission.      Herrmann — 

Klein.) 8vo,  5  00 

Machinery  of  Transmission  and  Governors.     (Herrmann — Klein.).  .8vo,  5  00 

Hydraulics  and  Hydraulic  Motors.     (Du  Bois.) 8vo,  5  00 

Wolff's  Windmill  as  a  Prime  Mover 8vo,  3  00 

Wood's  Turbines 8vo,  2  so 

MATERIALS   OF  ENGINEERING. 

Bovey's  Strength  of  Materials  and  Theory  of  Structures 8vo,  7  50 

Btirr's  Elasticity  and  Resistance  of  the  Materials  of  Engineering.     6th  Edition, 

Reset 8vo.  7  50 

Church's  Mechanics  of  Engineering 8vo,  6  00 

Johnson's  Materials  of  Construction Large  8vo,  6  00 

Keep's  Cast  Iron 8vo,  2  50 

Lanza's  Applied  Mechanics 8vo,  7  50 

Martens's  Handbook  on  Testing  Materials.     (HennLng.) 8vo,  7  50 

Merriman's  Text-book  on  the  Mechanica  of  Materials 8vo,  4  00 

•   Strength  of  Mater'als ". lamo,  i  00 

Metcalf's  SteeL     A  Manual  for  Steel-users i2mo.  a  00 

Smith's  Materials  of  Machines i2mo.  i  00 

Thurston's  Materials  of  Engineering 3  vols.,  Svo,  8  00 

Part   n. — Iron  and  Steel Svo,  3  50 

Part  in. — A  Treatise  on  Brasses,  Bronzes,  and  Other  Alloys  and  their 

Constituents 8vo  2  50 

Text-book  of  the  Ifaterials  of  Construction Svo,  5  00 

Wood's  Treatise  on  the  Resistance  of  Materials  and  an  Appendix  on  the 

Preservation  of  Timber .' Svo,  2  00 

Elements  of  Analytical  Mechanics Svo,  3  00 

Wood's  Rustless  Coatings:  Corrosion  and  Electrolysis  of  Iron  and  Steel. .  .Svo,  4  00 

STEAM-ENGINES  AND  BOILERS. 

Camot's  Reflections  on  the  Motive  Power  of  Heat.     (Thurston.) i2mo,  X  50 

Dawson's  "Engineering"  and  Electric  Traction  Pocket-book.  .i6mo,  mor.,  5  00 

Ford's  Boiler  Making  for  Boiler  Makers iSmo,  i  00 

13 


Ooss's  LocomotiTe  Sparks 8vo,  a  oo 

Hemenway's  Indicator  Practice  and  Steam-engine  Economy i2mo,  a  oo 

Hatton's  Mechanical  Engineering  of  Power  Plants 8vo,  5  00 

Heat  and  Heat-engines 8vo,  5  00 

Kent's  Steam-boiler  Economy 8vo,  4  00 

Kneass's  Practice  and  Theory  of  the  Injector 8vo  i  jo 

MacCord's  Slide-valves 8vo,  3  00 

Meyer's  Modem  Locomotive  Construction 4to.  10  00 

Peabody's  Manual  of  the  Steam-engine  Indicator i2mo,  i  50 

Tables  of  the  Properties  of  Saturated  Steam  and  Other  Vapors 8vo,  i  00 

Thermodynamics  of  the  Steam-engine  and  Other  Heat-engines 8vo,  5  00 

Valve-gears  for  Steam-engines 8vo,  2  50 

Peabody  and  Miller's  Steam-boilers 8vo,  4  00 

Pray'g  Twenty  Years  with  the  Indicator Large  8vo,  2  50 

Pupln's  Thermodynamics  of  Reversible  Cycles  in  Gases  and  Saturated  Vapors. 

(Osterberg.) i2mo.  i  2S 

Reagan's  Locomotives :  Simple,  Compound,  and  Electric i2mo,  2  50 

Rontgen's  Principles  of  Thermodynamics.     (Du  Bois.) 8vo,  5  00 

Sinclair's  Locomotive  Engine  Running  and  Management i2mo,  2  00 

Smart's  Handbook  of  Engineering  Laboratory  Practice i2mo,  2  50 

Snow's  Steam-boiler  Practice 8vo,  3  00 

Spangler's  Valve-gears 8vo,  2  50 

Notes  on  Thermodynamics i2mo,  1  00 

Spangler,  Greene,  and  Marshall's  Elements  of  Steam-engineering 8vo,  3  00 

Thurston's  Handy  Tables 8vo,  i  50 

Manual  of  the  Steam-engine 2  vols.  8vo,  10  00 

Part  I. — History,  Structuce,  and  Theory 8vo,  6  00 

Part  n. — Design,  Construction,  and  Operation 8vo,  6  00 

Handbook  of  Engine  and  BoHer  Trials,  and  the  Use  of  the  Indicator  and 

the  Prony  Brake 8vo  5  o» 

Stationary  Steam-engines 8vo,  2  50 

Steam-boiler  Explosions  in  Theory  and  in  Practice i2mo  1  50 

Manual  of  Steam-boilers , Their  Designs,  Construction,  and  Operation. 8vo,  5  00 

Weisbach's  Heat,  Steam,  and  Steam-engines.     (Du  Bois.) 8vo,  5  o« 

Whitham's  Steam-engine  Disign 8vo,  5  00 

Wilson's  Treatise  on  Steam-boilers.     (Flather.) i6mo,  2  50 

Wood's  Thermodynamics   Heat  Motors,  and  Refrigerating  Machines. . .  .8vo,  4  00 


MECHANICS    AND   MACHINERY. 


Barr's  Kinematics  of  Machinery 8vo,  2  50 

Bovey's  Strength  of  Materials  and  Theory  of  Structures 8vo,  7  50 

Chase's  The  Art  of  Pattern-making i2mo,  2  50 

ChordaL — Extracts  from  Letters i2mo,  2  00 

Church's  Mechanics  of  Engineering 8vo,  6  00 

Notes  and  Examples  in  Mechanics ; 8vo,  2  00 

Compton's  First  Lessons  in  Metal-working i2mo,  i  50 

Compton  and  De  Groodt's  The  Speed  Lathe i2mo,  1  50 

Cromwell's  Treatise  on  Toothed  Gearing i2mo,  1  50 

Treatise  on  Belts  and  Pulleys .  .      i2mo,  i  50 

Dana's  Text-book  of  Elementary  Mechanics  for  the  Use  of  Colleges  and 

Schools i2mo,  I  50 

Dingey's  Machinery  Pattern  Making i2mo,  2  00 

Dredge's   Record   of   the   Transportation   Exhibits  Building  of  the   World's 

Columbian  Exposition  of  1893 4to,  half  morocco,  5  00 

13 


Du  Bois's  Elementary  Principles  of  Mechanics : 

VoL     I. — Kinematics 8vo,  3  50 

Vol.   II. — Statics 8vo,  4  00 

Vol.  in. — Kinetics .8vo,  3  50 

Mechanics  of  Engineering.     VoL   I Small  4to,  7  50 

Vol.  II Small  4to,  10  00 

Durley's  Kinematics  of  Machines 8vo,  4  00 

Fitzgerald's  Boston  Machinist i6mo,  i  00 

Flather's  Dynamometers,  and  the  Measurement  of  Power i2mo,  3  00 

Rope  Driving i2mo,  3  00 

Goss's  Locomotive  Sparks 8vo  2  00 

Hall's  Car  Lubrication i2mo,  i  00 

Holly's  Art  of  Saw  Filing i8mo,  75 

♦  Johnson's  Theoretical  Mechanics i2mo,  3  00 

Statics  by  Graphic  and  Algebraic  Methods 8vo,  2  00 

Jones's  Machine  Design: 

Part  I. — Kinematics  of  Machinery 8vo,  i  50 

Part  II. — Form,  Strength,  and  Proportions  of  Parts 8vo,  3  00 

Kerr's  Power  and  Power  Transmission 8vo,  2  00 

Lanza's  Applied  Mechanics 8vo,  7  50 

MacCord's  Kinematics;  or,  Practical  Mechanism 8vo,  5  00 

Velocity  Diagrams 8vo,  i  50 

Maurer's  Technical  Mechanics 8vo,  4  00 

Meniman's  Text-book  on  the  Mechanics  of  Materials 8vo,  4  00 

*  Michie's  Elements  of  Analytical  Mechanics Svo,  4  00 

Reagan's  Locomotives:  Simple,  Compound,  and  Electric i2mo,  2  50 

Reid's  Course  in  Mechanical  Drawing Svo,  2  00 

Text-book  of  Mechanical  Drawing  and  Elementary  Machine  Design.  .8vo,  3  00 

Richards's  Compressed  Air lamo,  1  50 

Robinson's  Principles  of  Mechanism 8vo,  3  00 

Ryan,  Norris,  and  Hoxie's  Electrical  Machinery.     Vol.  I. Svo,  2  50 

Sinclair's  Locomotive-engine  Running  and  Management i2mo,  2  00 

Smith's  Press-working  of  Metals Svo,  3  00 

Materials  of  Machines i2mo,  i  00 

Spangler,  Greene,  and  Marshall's  Elements  of  Steam-engineering Svo,  3  00 

Thurston's  Treatise  on  Friction  and  Lost  Work  in  Machinery  and  Mill 

Work Svo,  3  00 

Animal  as  a  Machine  and  Prime  Motor,  and  the  Laws  of  Energetics.  i2mo,  i  00 

Warren's  Elements  of  Machine  Construction  and  Drawing Svo,  7  50 

Weisbach's    Kinematics    and    the  Power  of    Transmission.     (Herrmann — 

Klein.) •  -Svo,  5  00 

Machinery  of  Transmission  and  Governors.     (Herrmann — Klein.). Svo,  5  00 

Wood's  Elements  of  Analytical  Mechanics Svo,  3  00 

Principles  of  Elementary  Mechanics i2mo,  i  25 

Turbines 8vo,  2  50 

The  World's  Columbian  Exposition  of  1S93 4to»  i  00 

METALLURGY. 

Egleston's  Metallurgy  of  Silver,  Gold,  and  Mercury: 

VoL   I.— SUver 8vo,  7  50 

VoL   n.— Gold  and  Mercury Svo,  7  So 

**  Iles's  Lead-smelting.     (Postage  9  cents  additional.) i2mo,  2  50 

Keep's  Cast  Iron 8vo,  2  50 

Kunhardt's  Practice  of  Ore  Dressing  in  Europe Svo,  i  50 

Le  Chatelier's  High-temperature  Measurements.  (Boudouard — Burgess.) .  i2mo,  3  00 

Metcalf's  SteeL    A  Manual  for  Steel-users i2mo,  2  00 

Smith's  Materials  of  Machines i2mo,  i  00 

14 


Thurston's  Materials  of  Engineering.     In  Three  Parts 8vo,  8  oo 

Part   II. — Iron  and  Steel Svo,  3  50 

Part  III. — A  Treatise  on  Brasses.  Bronzes,  and  Other  Alloys  and   their 

Constituents Svo,  2  50 

Ulke's  Modem  Electroljrtic  Copper  Refining Svo,  3  00 

MINERALOGY. 

Barringer's  Description  of  Minerals  of  Commercial  Value.     Oblong,  morocco,  2  50 

Boyd's  Resources  of  Southwest  Virginia Svo,  3  00 

Map  of  Southwest  Virginia Pocket-book  form,  2  00 

Brush's  Manual  of  Determinative  Mineralogy.     (Penfield.) Svo,  4  00 

Chester's  Catalogue  of  Minerals Svo,  paper,  i  00 

Cloth,  I   25 

Dictionary  of  the  Names  of  Minerals  Svo,  3  50 

Dana's  System  of  Mineralogy Large  Svo,  half  leather,  12  50 

First  Appendix  to  Dana's  New  "System  of  Mineralogy.". ..  .Large  Svo,  i  00 

Text-book  of  Mineralogy • Svo,  4  00 

Minerals  and  How  to  Study  Them i2mo,  i  50 

Catalogue  of  American  Localities  of  Minerals Large  Svo,  i  00 

Manual  of  Mineralogy  and  Petrog^raphy i2mo,  2  00 

Eakle's  Mineral  Tables Svo,  i  25 

Egleston's  Catalogue  of  Minerals  and  Synonyms Svo,  2  50 

Hussak's  The  Determination  of  Rock-forming  Minerals.     (Smith.)  Small  Svo,  2  00 

Merrill's  Non-metallic  Minerals:  Their  Occurrence  and  Uses Svo,  4  00 

*  Penfield's  Notes  on  Determinative  Mineralogy  and  Record  of  Mineral  Tests. 

Svo,  paper,  o  50 
Rosenbusch's   Microscopical   Physiography   of   the   Rock-making   Minerals. 

(Iddings.) Svo,  5  00 

•  Tillman's  Text-book  of  Important  Minerals  and  Docks Svo,  2  00 

Williams's  Manual  of  Lithology Svo,  3  00 

MIKING. 

Beard's  Ventilation  of  Mines i2mo,  2  50 

Boyd's  Resources  of  Southwest  Virginia Svo,  3  00 

Map  of  Southwest  Virginia Pocket-book  form,  2  00 

•  Drinker's  Tunneling,  Explosive  Compounds,  and  Rock  Drills. 

4to,  half  morocco,  25  00 

Eissler's  Modem  High  Explosives Svo,  4  00 

Fowler's  Sewage  Works  Analsrses i2mo,  2  00 

Goodyear's  Coal-mines  of  the  Western  Coast  of  the  United  States i2mo,  2  50 

Ihlseng's  Manual  of  Mining. , Svo,  4  00 

♦*  Iles's  Lead-smelting.     (Postage  gc.  additionaL) i2mo,  2  50 

Kunhardt's  Practice  of  Ore  Dressing  in  Europe Svo,  i  50 

O'DriscoU's  Notes  on  the  Treatment  of  Gold  Ores Svo,  2  00 

*  Walke's  Lectures  on  Explosives Svo,  4  00 

Wilson's  Cyanide  Processes i2mo,  1  so 

Cblorination  Process i2mo,  i  50 

Hydraulic  and  Placer  Mining i2mo,  2  00 

Treatise  on  Practical  and  Theoretical  Mine  Ventilation i2mo  i  25 

SANITARY  SCIENCE. 

Copeland's  Manual  of  Bacteriology.     (In  preparation.) 

Folwell's  Sewerage.     (Designing,  Construction  and  Maintenance.; Svo,  3  00 

Water-supply  Engineering Svo,  4  00 

Fuertes's  Water  and  PubUc  Health i2mo,  i  50 

Water-filtration   Works I2m0t  2  50 

15 


Gerhard's  Guide  to  Sanitary  House-inspection .  i6mo,  i  oo 

Goodrich's  Economical  Disposal  of  Town's  Refuse Demy  8vo,  3  50 

Hazen's  Filtration  of  Public  Water-supplies 8vo,  3  00 

Kiersted's  Sewage  Disposal i2mo,  i  25 

Leach's  The  Inspection  and  Analysis  of  Food  with  Special  Reference  to  State 

Control.     {In  preparation.) 
Mason's    Water-supply.     (Considered   Principally   from   a    Sanitary   Stand- 
point.)    3d  Edition,  Rewritten 8vo,  4  00 

Examination  of  Water.     (Chemical  and  BacteriologicaL) i2mo,  i   25 

Merriman's  Elements  of  Sanitary  Engineering , Svo,  2  00 

Nichols's  Water-supply.     (Considered  Mainly  from  a  Chemical  and  Sanitary 

Standpoint.)     (1883.) Svo.  2  50 

Ogden's  Sewer  Desig^n i2mo,  2  00 

Prescott  and  Winslow's  Elements  of  Water  Bacteriology,  with  Special  Reference 

to  Sanitary  Water  Analysis i2m0j  i  25 

*  Price's  Handbook  on  Sanitation i2mo,  i  50 

Richards's  Cost  of  Food.    A  Study  in  Dietaries i2mo,  i  00 

Cost  of  Living  as  Modified  by  Sanitary  Science i2mo,  i  00 

Richards  and  Woodman's  Air,  Water,  and  Food  from  a  Sanitary  Stand- 
point   Svo,  3  00 

♦  Richards  and  Williams'3  The  Dietary  Computer Svo,  i  50 

Rideal's  Sewage  and  Bacterial  Purification  of  Sewage Svo,  3  50 

Tumeaure  and  Russell's  Public  Water-supplies Svo,  5  00 

Whipple's  Microscopy  of  Drinking-water Svo,  3  50 

Woodhull's  Notes  and  Military  Hygiene i6mo,  1  50 


MISCELLANEOUS. 

Barker's  Deep-sea  Soundings Svo,  2  00 

Emmons's  Geological  Guide-book  of  the  Rocky  Mountain  Excursion  of  the 

International  Congress  of  Geologists Large  Svo  i  50 

Ferrel's  Popular  Treatise  on  the  Winds Svo  4  00 

Haines's  American  Railway  Management i2mo,  2  50 

Mott's  Composition.'Digestibility,  and  Nutritive  Value  of  Food.   Mounted  chart,  i  25 

Fallacy  of  the  Present  Theory  of  Sound i6mo  1  00 

Ricketts's  History  of  Rensselaer  Polytechnic  Institute,  1824-1894.  Small  Svo,  3  00 

Rotherham's  Empnasized  New  Testament ;  .Large  Svo,  2  00 

Steel's  Treatise  on  the  Diseases  of  the  Dog Svo,  3  50 

Totten's  Important  Question  in  Metrology Svo  2  50 

The  World's  Columbian  Exposition  ot  1893 4to,  i  00 

Worcester  and  Atkinson.     Small  Hospitals,  Establishment  and  Maintenance, 
and  Suggestions  for  Hospital  Architecture,  with  Plans  for  a  Small 

Hospital i2mo,  r  25 


HEBREW  AND  CHALDEE  TEXT-BOOKS. 

Green's  Grammar  of  the  Hebrew  Language Svo,  3  00 

Elementary  Hebrew  Grammar i2mo,  i  25 

Hebrew  Chrestomathy Svo,  2  00 

Gesenius's  Hebrew  and  Chaldee  Lexicon  to  the  Old  Testament  Scriptures. 

(Tregelles.) Small  4to,  half  morocco,  s  00 

Letteris's  Hebrew  Bible - Svo,  2  25 

16 


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